Apparatuses and methods for electrochemically modifying the retention of species on chromatography material

ABSTRACT

An apparatus and method for electrochemically modifying the retention of a species on a chromatography material is disclosed. The apparatus comprises a housing having an effluent flow channel adapted to permit fluid flow therethrough. The effluent flow channel comprises chromatography material. The apparatus further comprises first and second electrodes positioned such that at least a portion of the chromatography material is disposed between the first and second electrodes, and fluid flow through the apparatus is between, and in contact with, the first and second electrodes.

CROSS-REFERENCE TO PRIOR APPLICATIONS

This application is a continuation of and claims priority to U.S.application Ser. No. 11/131,864 filed on May 18, 2005, which is acontinuation of U.S. application Ser. No. 10/427,812 filed on May 1,2003, which is a continuation of U.S. application Ser. No. 09/689,176filed on Oct. 11, 2000 (now U.S. Pat. No. 6,558,551 issued on May 6,2003), which is a continuation of U.S. application Ser. No. 09/561,631filed on May 2, 2000 (now U.S. Pat. No. 6,613,235 issued on Sep. 2,2003), which is a continuation of U.S. application Ser. No. 08/609,171filed on Mar. 1, 1996 (now U.S. Pat. No. 6,093,327 issued on Jul. 25,2000), which is a continuation-in-part of U.S. application Ser. No.08/486,210 filed on Jun. 7, 1995 (now abandoned), which is acontinuation-in-part of U.S. application Ser. No. 08/399,706 filed onMar. 3, 1995 (now abandoned).

An Appendix consisting of 67 sheets is included in this application. TheAppendix contains material which is subject to copyright protection. Thecopyright owner has no objection to the facsimile reproduction of anyone of the sheets of the Appendix, as it appears in the Patent andTrademark patent files or records, but otherwise reserves all copyrightrights to this material whatsoever. The pages of the Appendix areincorporated herein by reference as though fully set forth herein.

FIELD OF THE INVENTION

This invention relates to chromatography columns, apparatuses, andmethods. In particular, the columns of the present invention can be usedas the separation column in electrocution chromatography and are alsoadaptable for use as a self-regenerating suppressor in suppressed ionchromatography. The columns of the present invention may also be used toseparate a wide range of compounds on both an analytical and preparativescale.

BACKGROUND OF THE INVENTION

A. Single Column Ion Chromatography

Single Column Ion Chromatography (SCIC) is a method of ion analysis inwhich ions are separated in an ion exchange column (e.g., separatorcolumn) and subsequently measured by a conductivity detector connecteddirectly to the separator column. In SCIC, special ion exchange resinsof low capacity, and eluants with either much higher or much lowerequivalent conductance than the ions being measured must be employed. Inion chromatography, sample ions generate a signal at a conductivitydetector. The signal is proportional to the sample ion concentration andis the difference in equivalent conductance between the sample ion andthe eluant ion. SCIC sensitivity is limited by the difference inequivalent conductance between the sample ions and the eluant ions. Thissensitivity is adequate and even preferred for some sample types,especially for cationic samples, where the difference in equivalentconductance between the sample and eluant ions is very large. However,for many other samples, particularly anionic samples, where thedifference in equivalent conductance between the sample ions and eluantions is small, sensitivity can be greatly increased by a second andpreferred type of ion analysis called chemically suppressed ionchromatography (SIC).

B. Suppressed Ion Chromatography (SIC)

Suppressed ion chromatography (SIC) is a form of commonly practiced ionanalysis characterized by the use of two ion-exchange columns in seriesfollowed by a flow through conductivity detector. The first column,called the separation column, separates the ions of an injected sampleby elution of the sample through the column using an electrolyte as aneluant, i.e., usually dilute base or acid in deionized water. The secondcolumn, called the “suppressor” or “stripper”, serves two purposes.First, it lowers the background conductance of the eluant to reducenoise. Second, it enhances the overall conductance of the sample ions.The combination of these two factors significantly enhances the signalto noise ratio, thus increasing sensitivity.

This technique is described in more detail in U.S. Pat. Nos. 3,897,213,3,920,397, 3,925,019 and 3,926,559. In addition, suitable ion exchangepackings for the separation column are described in detail in U.S. Pat.Nos. 3,966,596, 4,101,460 and 4,119,580. A detailed description of ionchromatography is additionally provided in Small et al., “Proceedings ofan International Conference on the Theory and Practice of Ion Exchange,”University of Cambridge, U.K., July, 1976; and also, Small et al.,“Novel Ion Exchange Chromatographic Method Using ConductimetricDetection”, Analytical Chemistry, Vol. 47, No. 11, September 1975, pp.1801 et seq. The foregoing patents and literature publications are fullyincorporated herein by reference.

C. Gradient Elution Technology

To separate or elute sample ions retained on an ion-exchange column, aneluant containing co-ions of the same charge of the sample ions isrouted through the separation column. The sample co-ions in the eluantpartially displace the sample ions on the ion-exchange column, whichcause the displaced sample ions to flow down the column along with theeluant. Typically, a dilute acid or base solution in deionized water isused as the eluant. The eluant is typically prepared in advance androuted through the column by either gravity or a pump.

Rather than using a homogenous eluant throughout the separation process,it is sometimes advantageous to use a gradient eluant, i.e., an eluantwherein the concentration of one or more components changes with time.Typically, the eluant starts at a weak eluting strength (e.g. a lowconcentration of the sample co-ions) and gets stronger (e.g. a higherconcentration of the sample co-ions) during the separation process. Inthis way, easily eluted ions are separated during the weaker portion ofthe gradient, and ions that are more difficult to elute are separatedduring the stronger portion of the gradient. The eluant concentrationchanges during the gradient and suppressing or balancing the concurrentchange in background conductance is required so the sample signal may bediscriminated from the background signal. An example of such gradientelution techniques are disclosed in U.S. Pat. Nos. 4,751,189 and5,132,018, the entire disclosures of which are incorporated herein byreference.

While the above patents utilize solutions prepared in advance to form agradient eluant, U.S. Pat. No. 5,045,204 to Dasgupta et al. useselectrochemical methods to generate a high purity eluant stream that mayflow directly to the separation column as it is produced, and which maybe generated as a gradient. In the Dasgupta patent, a product channel isdefined by two permselective membranes and is fed by a source ofpurified water.

One of the permselective membranes only allows the passage of negativelycharged hydroxide ions, which are generated on the side of this membraneopposite the product channel by the electrolysis of water at a cathode.The hydroxide ions are driven by an electric field through the membraneinto the product channel in an amount corresponding to the strength ofthe electric field. The other permselective membrane only allows thepassage of positively charged ions. On the side of this membraneopposite the product channel there is a source channel, which iscontinuously fed with a NaOH solution and in which an anode ispositioned. The Na⁺ ions are driven by the electric field through themembrane into the product channel in an amount corresponding to thestrength of the electric field. By this process, a high purity sodiumhydroxide (NaOH) solution is produced. This solution may be used as theeluant for a chromatography column, and the concentration of this eluantmay be varied during the chromatographic separation by varying thestrength of the electric field, thereby generating a gradient eluant.

The foregoing methods of elution ion chromatography suffer from certaindisadvantages, however. Among these disadvantages is that an outsidesource of eluant or eluant counter-ions is required. Also, after elutingthe sample ions from the chromatography column, all of these eluantsrequire suppression in order to provide an accurate quantitativeanalysis of the sample ions. Finally, in general practice, all of theabove methods of eluting are only applicable to one of either cation oranion sample ions within a single sample run. If one wishes to analyzeboth the cations and anions from a single sample, two chromatographicseparations must be performed using either two apparatuses and twodistinct eluants, or a single instrument with two or more columns andcomplex switching valves.

D. Prior Suppressor Technology

Chemical suppression for IC serves two purposes. First, it lowers thebackground conductance of the eluant to reduce baseline noise. Second,it enhances the overall conductance of the sample ions to increase thesignal. The combination of these two factors significantly enhances thesignal-to-noise ratio, and increase the detectivity of the sample ions.For example, in anion analysis, two ion-exchange reactions take place ina suppressor column when the eluant comprises sodium hydroxide and theion exchange packing material in the suppressor column comprisesexchangeable hydronium ions:

-   1) Eluant: NaOH+Resin−SO₃ ⁻H⁺→Resin−SO₃ ⁻Na⁺+H₂O-   2) Analyte: NaX+Resin−SO₃ ⁻H⁺→Resin−SO₃ ⁻Na⁺+HX where X=anions (Cl⁻,    NO₂ ⁻, Br⁻, etc.)

The relatively high conductivity sodium hydroxide eluant is converted tothe relatively low conductivity water when the sodium ions from theeluant displace the hydronium ions on the ion exchange packing materialin the suppressor. The sample anions are converted from their salt forminto their more conductive acid form by exchanging their counter-ionsfor hydronium ions in the suppressor. The eluant is preferably asolution of any salt that forms a weakly conductive acid after goingthrough the suppressor. Examples of such eluants in anion analysisinclude sodium hydroxide, sodium carbonate, or sodium tetraboratesolutions.

Various suppressor devices that operate on the above principles havebeen used for IC. These include:

1. Packed-Bed Suppressors

Packed-Bed Suppressors were introduced in about 1973 (see, for example,U.S. Pat. Nos. 3,918,906, 3,925,019, 3,920,397, 3,926,559, 4,265,634,and 4,314,823, the entire disclosures of which are incorporated hereinby reference). These suppressors consist of large columns containingstrong acid cation-exchange resins in hydronium form (for anionanalysis). In order to house enough resin, these columns are very large(i.e., 250 mm×7.8 mm). However, these columns have a large dead volume,which causes considerable peak dispersion and broadening. This, in turn,results in a loss of chromatographic efficiency. Moreover, after severalhours of operation, the resin bed becomes exhausted (all the hydroniumions on the exchange sites are replaced by the sample and the eluantcounterions). The suppressor column must then be taken off-line andregenerated by flushing the column with an acid to regenerate thehydronium ion exchange sites in the resin bed. The regeneration of thesuppressor column, of course, is time consuming and interrupts theanalysis.

Another disadvantage of these packed bed suppressors is that weaklyionized species such as organic acids can penetrate the protonatedcation exchange sites and interact by inclusion within the resin bed.This causes variable retention times and peak areas as the suppressorbecomes exhausted.

Also, some ions can undergo chemical reactions in the suppressor. Forexample, nitrite has been shown to undergo oxidation in these prior artpacked bed suppressors leading, to variable recovery and poor analyticalprecision.

2. Hollow-Fiber Membrane Suppressors

In about 1982, hollow fiber membrane suppressors were introduced (see,for example, U.S. Pat. Nos. 4,474,664 and 4,455,233, the entiredisclosures of which are incorporated herein by reference). Hollow fibermembrane suppressors were designed to overcome the drawbacks of thepacked bed suppressors. The hollow fiber membrane suppressors consist ofa long, hollow fiber made of semi-permeable, ion-exchange material.Eluant passes through the hollow center of the fiber, while aregenerating solution bathes the outside of the fiber. Suppressor ionscross the semi-permeable membrane into the hollow center of the fiber,and suppress the eluant. The regenerating solution provides a steadysource of suppressor ions, allowing continual replacement of thesuppressor ions as they pass to the eluant flow channel in the hollowcenter of the fiber. The main advantage of the hollow fiber design isthat the chromatography system can be continuously operated becausethere is no need to take the suppressor off-line for regeneration, as isthe case with the packed bed suppressors.

However, the hollow fiber design introduced new problems. The smallinternal diameter of the fibers reduces the surface area available forion exchange between eluant and the regenerant. This limits thesuppression capability of the hollow fiber suppressors to low flow ratesand low eluant concentrations. Additionally, because the fiber is bathedin the regenerant solution, the counterion of the suppressor ions canleak into the eluant channel, and cause higher background conductivityand baseline noise at the detector.

3. Flat-Sheet Membrane Suppressors

Flat-sheet membrane suppressors were introduced in about 1985 (see, forexample, U.S. Pat. Nos. 4,751,189 and 4,999,098, the entire disclosuresof which are incorporated herein by reference). In these suppressors,the ion exchange tubing in the hollow-fiber suppressor is replaced withtwo flat semi-permeable ion exchange membranes sandwiched in betweenthree sets of screens. The eluant passes through a central chamber whichhas ion exchange membrane sheets as the upper and lower surfaces. Thevolume of the eluant chamber is very small, so band broadening isminimal. Since the membrane is flat, the surface area available forexchange between the sample counterions and the suppressor ions in theregenerant is greatly increased. This increases the suppression capacityallowing high flow rates, high eluant concentration, and gradientanalyses. Preferably, the regenerant flows in a direction counter to thesample ions over the outer surfaces of both membranes, providing aconstant supply of suppressor ions.

A major drawback, however, of membrane suppressors is that they requirea constant flow of regenerant to provide continuoussuppression/operation. This consumes large volumes of regenerant andproduces large volumes of chemical waste, significantly increasingoperating cost. An additional pump or device is required to continuouslypass the regenerant through the suppressor, increasing the instrument'scomplexity and cost while reducing reliability. Also, organic compoundscan irreversibly adsorb onto the hydrophobic ion-exchange membrane,reducing its efficiency to the point where it requires replacement(membranes are typically replaced every six months to two years).Finally, the membranes are very thin and will not tolerate muchback-pressure. Thus, membrane rupture is a concern anytime downstreambackpressure increases due to blockages.

4. Solid Phase Chemical Suppressor (SPCS)

Alltech Inc., the assignee of the present application, developed solidphase chemical suppressors (SPCS) in about 1993, which were essentiallyan improved version of the original packed bed suppressors. Problemsassociated with the original packed bed suppressors, such as bandbroadening, variable retention time and peak area, and the oxidation ofnitrite in the suppressor, were greatly reduced. The Alltech SPCS usesdisposable cartridges containing ion exchange packing materialcomprising suppressor ions as the suppressor device. The inexpensivecartridges are simply discarded and replaced with a new cartridge whenthe suppressor ions are exhausted. Thus, no regeneration is required,thereby eliminating the need for expensive or complex systems forregenerating suppressor ions.

In Alltech's SPCS system, a 10-port switching valve and two disposablesuppressor cartridges are typically employed. The effluent from theanalytical column flows through one cartridge at a time. While onecartridge is being used, the suppressed detector effluent (typicallywater or carbonic acid) flows through the other suppressor cartridge topre-equilibrate the cartridge. This reduces the baseline shift due toconductance change when the valve is switched to the other suppressorcartridge. When all the suppressor ions from one cartridge are replacedby the eluant and sample counterions, the valve is switched, placing thesecond cartridge in the active position, and the exhausted suppressorcartridge is replaced. This allows continuous operation. However, theAlltech system still requires someone to switch the valve manually whenthe first cartridge is exhausted. Each cartridge typically providesbetween 6 to 9 hours of operation, and thus fully unattended orovernight operation might not be possible in certain applications withthe Alltech SPCS system.

5. Electrochemical Suppression

Electrochemical suppressors were introduced in about 1993. Thesesuppressors combine electrodialysis and electrolysis in a flat-sheetmembrane suppressor column similar to those described under headingsection 3 above (see U.S. Pat. Nos. 4,459,357 and 5,248,426, the entiredisclosures of which are incorporated herein by reference).

For example, U.S. Pat. No. 5,248,426 to Stillian et al. discloses asuppressor which contains a central chromatography effluent flow channelbordered on both sides by ion exchange membranes with exchangeable ionsof the opposite charge of the sample ions. On the side of each membraneopposite the effluent flow channel are first and second detectoreffluent flow channels. The sample ions and eluant are routed throughthe chromatography effluent flow channel, and the water-containingdetector effluent is routed through the detector effluent flow channelsin the suppressor. An electrode is positioned in both of the detectoreffluent flow channels.

By energizing the electrodes, an electrical potential is generated inthe suppressor transverse to the liquid flow through the chromatographyflow channel. When the water-containing detector effluent contacts theenergized electrodes, it undergoes electrolysis. In anion analysis forexample, the suppressor hydronium ions generated at the anode in a firstdetector-effluent channel are transported across the ion exchangemembrane into the chromatography effluent flow channel, where theycombine with the sample anions to form the highly conductive acids ofthe sample anions. The suppressor hydronium ions also combine with thehydroxide ions in the eluant (in anion analysis) to convert the eluantinto the relatively non-conductive water. At the same time, the eluantand sample counterions are transported from the chromatography effluentchannel across the ion exchange membrane into a second detector effluentflow channel where they combine with the hydroxide ions generated by theelectrolysis of the water-containing detector effluent at the cathode inthe second detector effluent flow channel. The resulting bases of theeluant counterions are then routed to waste.

Thus, the electric field generated in the suppressor column disclosed inStillian et al. simultaneously generates suppressor ions and promotesion-flow between the electrodes in a direction transverse to the fluidflow through the suppressor. The mass transport of ions is across afirst ion exchange membrane from a first detector effluent flow channelto the chromatography effluent flow channel, and across a second ionexchange membrane to a second detector effluent flow channel.

Although the electrochemical suppressor device disclosed in Stillian etal. offers certain advantages (i.e. no separate regenerant source isrequired), it still suffers from certain disadvantages. Irreversibleadsorption of organic components and membrane breakage under pressuremay still occur in the apparatus and method disclosed in Stillian. Also,the method of electrochemical suppression disclosed in Stillian can onlybe used to analyze solely anions or solely cations in any one sample.Finally, the Stillian method does not work well with electroactiveeluants or organic solvents. Electroactive eluants, such as hydrochloricacid, commonly employed as an eluant for cation analysis, undergoelectrochemical reaction in the suppressor producing by-products thatdamage the membrane. Also, certain organic eluant components such asmethanol undergo electrochemical reaction in the electrochemicalsuppressor producing by-products that are conductive and which interferewith the detection of sample ions. Such electroactive eluant systems maynot be effectively employed in the Stillian method.

The column, apparatuses, and methods of the present invention reduce oravoid many of the foregoing problems.

SUMMARY OF THE INVENTION

In one aspect of the present invention, the foregoing disadvantages areovercome. The column of the present invention can be used in apparatusesand methods for generating an eluant in-situ that does not require anoutside source of sodium or other electrolytes. Additionally, the columnof the present invention can be used in apparatuses and methods thatgenerate a self-suppressing eluant and, therefore, a second suppressorcolumn is not required. Moreover, the column of the present inventioncan be adapted for use in apparatuses and methods for analyzing bothcations and anions in a single sample run.

In one embodiment of the present invention, a housing is provided. Thehousing has an effluent flow channel adapted to permit fluid flowthrough the housing. The housing further contains chromatography packingmaterial disposed in the effluent flow channel. The housing alsocontains first and second electrodes which are positioned such that atleast a portion of the chromatography packing material is disposedbetween the first and second electrodes, and fluid flow through thehousing is from one of the first or second electrodes to the other.

In another aspect of the invention, an apparatus for electrochemicallymodifying the retention of a species on chromatography material isprovided. The apparatus has a housing, which comprises an effluent flowchannel. The effluent flow channel comprises chromatography material,and the effluent flow channel is adapted to permit fluid flowtherethrough. The apparatus further comprises a first electrode and asecond electrode. These first and second electrodes are positioned suchthat at least a portion of the chromatography material is disposedbetween the first and second electrodes, and the fluid flow through theeffluent flow channel is between, and in contact with, the first andsecond electrodes. Also, the apparatus further comprises a power sourceconnected to the first and second electrodes.

In yet another aspect of the invention, a method of electrochemicallymodifying the retention of a compound or species on a chromatographymaterial is provided. According to this method, an effluent flow channelis provided comprising a stationary phase containing chromatographymaterial on which the compound or species is retained. A first and asecond electrode are also provided, and the electrodes are positionedsuch that at least a portion of the chromatography material is disposedbetween the first and second electrodes. A mobile phase comprising aneluant is further provided. The eluant is flowed between, and in contactwith, the first and second electrodes thereby electrochemicallymodifying the eluant. The modified eluant is flowed to thechromatography material, which modifies the retention of the compound orspecies on the chromatography material.

The foregoing housing and apparatus may be used as a chromatographycolumn, a self-regenerating suppressor, and in various chromatographyapparatuses according to various embodiments of the present invention.The apparatus of the present invention may also be used in variousmethods of separating ions, proteins, and other compounds. The apparatusof this invention may also be used in methods of generating a highpurity eluant, and for generating a gradient in gradient elutionchromatography.

These and other advantages of the invention, as well as the inventionitself, will be best understood with reference to the attached drawings,a brief description of which follows, along with the detaileddescription of the invention provided herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a perspective view of a preferred column of the presentinvention.

FIG. 2 is an exploded view of the column illustrated in FIG. 1.

FIG. 3 is a cross-sectional view of the column illustrated in FIG. 1.

FIG. 4 is a schematic view of a chromatography apparatus for use in amethod of electroelution chromatography.

FIGS. 5A and 5B are schematic views of the chromatography column of FIG.1 showing ion exchange when the column is used in a method ofelectroelution chromatography.

FIG. 6 is a schematic view of a chromatography apparatus for use in amethod of electroelution chromatography.

FIG. 7 is an illustrative chromatogram using the chromatographyapparatus depicted in FIG. 6 in a method of electroelutionchromatography where anions and cations in the same sample are detected.

FIGS. 8A-8D are schematic views of chromatography apparatuses where thecolumn of FIG. 1 is used as a solid phase chemical suppressor.

FIGS. 9A and 9B are schematic views of a chromatography apparatus wherethe column of FIG. 1 is used as a solid phase chemical suppressor.

FIGS. 10A and 10B are schematic views of the chromatography apparatusillustrated in FIGS. 9A and 9B, except that a different valve scheme isshown.

FIGS. 11A and 11B are schematic views of a chromatography apparatuswhere two columns of the present invention are used as an eluantgenerating column to generate a high purity eluant, and as a solid phasechemical suppressor, respectively.

FIG. 11C is a schematic view of a chromatography apparatus where thecolumn of FIG. 1 is used as a solid phase chemical suppressor.

FIGS. 12A and 12B are schematic views of a chromatography apparatuswhere the column of FIG. 1 is used in a hydrophobic suppressor unit.

FIG. 13 is a graph illustrating the suppression capacity of a columnaccording to one embodiment of the present invention.

FIG. 14 is a graph illustrating the suppression life of a columnaccording to one embodiment of the present invention.

FIG. 15 is a chromatogram of a sample containing ions using a columnaccording to one embodiment of the present invention.

FIG. 16 is a chromatogram of a sample containing ions using a columnaccording to one embodiment of the present invention.

FIG. 17 is a chromatogram of a sample containing ions using a columnaccording to one embodiment of the present invention.

FIG. 18 is a chromatogram of a sample containing ions using a columnaccording to one embodiment of the present invention.

FIG. 19 is a chromatogram of a sample containing ions using a columnaccording to one embodiment of the present invention.

FIG. 20 is a chromatogram of a sample containing ions using a columnaccording to one embodiment of the present invention.

FIG. 21 is a chromatogram of a sample containing ions using a columnaccording to one embodiment of the present invention.

FIG. 22 is a chromatogram of a sample containing ions using a columnaccording to one embodiment of the present invention.

FIGS. 23( a)-(c) is a flowchart for the computer program used in onepreferred aspect of the invention.

FIG. 24 is a cross-sectional view of a suppressor column according toone aspect of the present invention.

DETAILED DESCRIPTION OF THE DRAWINGS AND PRESENTLY PREFERRED EMBODIMENTS

The preferred column of the present invention is especially adapted foruse in chromatography apparatuses and methods. With reference to FIGS.1-3, the preferred chromatography column comprises a housing 1. Thehousing 1 consists of female end fitting 2 and male end fitting 3. Thefemale end fitting and the portion of the male end fitting other thanarm 4 are preferably made from a conductive material. Preferably, thesepieces are made from titanium or stainless steel coated with inertmaterial. The male end fitting 3 has a threaded arm 4 secured thereto.The arm 4 of the male end fitting 3 further comprises a cavity 4 b. Thethreaded arm is preferably made from a non-conductive, water proofplastic material. The arm 4 of male end fitting 3 is most preferablymade from polyetheretherketone (PEEK). The female end fitting 2 also hasa cavity 6. The cavity 6 is preferably threaded and adapted to receivethe threaded arm 4 of male end fitting 3.

Housing 1 is easily assembled by releasably securing arm 4 of male endfitting 3 in the cavity 6 of the female end fitting 2. This isaccomplished by simply screwing the threaded arm 4 of the male endfitting 3 into the cavity 6 of female end fitting 2. Similarly, thehousing 1 may be just as easily dismantled by unscrewing the threadedarm 4 of male end fitting 3 from the cavity 6 of female end fitting 2.When the housing 1 is assembled, the cavity 4 b of the arm 4 of male endfitting 3 provides part of an effluent flow channel 9, which is adaptedto permit fluid flow through the housing 1.

The male end fitting 3 has an opening 5, which is adapted to permitfluid to enter or exit the housing 1. The female end fitting 2 also hasan opening 7 which is adapted to permit fluid to enter or exit thehousing 1. Preferably, openings 5 and 7 are threaded for easy connectionto fluid lines in the chromatography apparatuses and methods describedherein. Extending from the opening 5 of male end fitting 3 to opening 7of female end fitting 3 when the housing 1 is assembled is the effluentflow channel 9.

The housing 1 further preferably comprises first and second electrodes11 and 13. Preferably, first and second electrodes 11 and 13,respectively, are positioned at opposite ends of the effluent flowchannel 9, and are positioned such that the fluid flow through thehousing 1 is from one of the first or second electrodes to the other. Inthe most preferred embodiment, the electrodes 11 and 13 are positionednear the openings 5 and 7 of the male end fitting 3 and female 2 endfitting, respectively, of the housing 1.

The housing 1 further comprises chromatography packing material 15disposed within the effluent flow channel 9. The chromatography packingmaterial 15 is selected as discussed with respect to the variousembodiments discussed below. Preferably, at least a portion of thechromatography packing material is disposed between the first and secondelectrodes. However, in an alternative embodiment of the presentinvention, those skilled in the art will appreciate that, instead ofusing chromatography packing material 15, the effluent flow channel 9may be defined by chromatography material (not shown). For example,chromatography material (not shown) may be coated on the wall 9 a of theeffluent flow channel 9.

Alternatively, the wall 9 a of effluent flow channel 9 may comprisechromatography material such as a hollow tubing containingchromatography stationary phases (not shown). One such material suitablefor use in this alternate embodiment is Nafion® available from PermaPure, Toms River, N.J. In view of the foregoing, those in the art willappreciate, the term “chromatography material” as used herein is meantto include chromatography packing material 15 (such as those materialsdiscussed herein), coatings of chromatography material containingchromatography stationary phases (not shown) coated on the wall 9 aproximate to the effluent flow channel 9, hollow tubing containingchromatography stationary phases, as well as other stationary phasescommonly used in chromatography.

In a preferred embodiment, the electrodes 11 and 13 are flow-throughelectrodes. By flow-through electrodes, it is meant that the electrodesallow the sample ions and eluant to flow therethrough. The electrodesare preferably made from carbon, platinum, titanium, stainless steel orany other suitable conductive, non-rusting material. The preferredflow-through electrodes are sufficiently porous to allow the sample ionsand eluant to flow therethrough, but sufficiently nonporous tophysically retain the packing material 15 disposed in the effluent flowchannel 9. The most preferred electrodes are made of platinum coatedtitanium, ruthenium oxide coated titanium, titanium nitride coatedtitanium, gold, or rhodium with an average pore size of between 0.1 μmand 100 μm.

In a preferred aspect of the invention, the flow-through electrodescomprise an annular surface 13 a surrounding an inner meshed surfacecomprising a frit 13 b. Preferably, only the annular surface 13 a iselectroactive; the inner frit surface 13 b being made from anon-electroactive material. The annular surface 13 a may be made fromany of the electroactive materials described above. The inner fritsurface 13 b is preferably made from PAT™ available from Systec(Minneapolis, Minn.), which is a non-electroactive alloy of TEFLON andPEEK. The foregoing electrode structure provides certain advantages whenthe eluant comprises an organic substance. Methanol, for example, isconverted to formic acid if it comes into contact with a charged surfaceof the electrode. Thus, if the eluant comprises methanol, it would beconverted to formic acid upon contacting the frit surface of theelectrode if the frit was made from an electroactive material. Such aresult is undesirable in that, among other things, the formic acidby-product could interfere with the analysis. Making the frit surfacefrom a non-electroactive material minimizes the oxidation of methanol,which reduces undesirable by-products when the eluant comprises organicsubstances such as methanol.

In a most preferred aspect of the invention, the electroactive surfacesof the electrodes 11 and 13 are coated with a Nafion® coating. Nafion™is a perfluorinated, hydrophilic, proton conducting ion exchange polymerthat exhibits relatively high thermal stability and is not detrimentalto the kinetics of electrochemical processes. Further informationconcerning the Nafion™ coating may be obtained from William T.Callaghan, Manager Technology Commercialization JPL301350 4800 Oak GroveDrive, Pasadena, Calif. 91104. When making such an inquiry refer toNPO19204, Vol. 19, No. 6, NASA Tech. Briefs, p. 66. The foregoingbrochure is incorporated herein by reference. The benefits of coatingthe electrode with Nafion™ is that, as presently understood, the voltagerequired to obtain a given current may drop by as much as twenty percentwhen the electrodes 11 and 13 are coated with Nafion™.

The electrodes 11 and 13 are connected to an electrical power source(not shown) via a spade lug (not shown) which is secured in lugreceptacles 5 a and 5 b of female end fitting 2 and male end fitting 3,respectively, by a screw or some other similar means. When the powersource (not shown) is turned on, an electric current, caused by iontransport, is established from one of the first or second electrodes tothe other across the chromatography packing material 15 when the columnis in use. Preferably, the electric current follows along a path that isparallel to fluid flow through the column. Most preferably, the currentis a constant current.

As described more fully below, the foregoing column can be used invarious apparatuses and methods of electroelution chromatography. Thecolumn of the present invention may also be advantageously used as aself-regenerating chemical suppressor. In addition, the column of thepresent invention can be used in a variety of other applications aswell, which are also described herein.

A. Electroelution Elution Chromatography

FIGS. 4-6 illustrate the preferred column of the present inventionespecially adapted for use in preferred apparatuses and methods forseparating, detecting, and analyzing sample ions by electroelutionchromatography. As used herein, the term “electroelution chromatography”means eluting sample components from a chromatographic column byelectrochemically generating or modifying the mobile phase. In otherwords, the mobile phase is generated or modified within or prior toentering the column by electrochemical action on the eluant.

FIG. 4 is a schematic view of an apparatus for ion analysis byelectroelution chromatography using the preferred column of the presentinvention. The present embodiment is discussed with respect to thedetection of anions in a sample. However, as discussed below, thisembodiment may be modified for cation analysis, or for the analysis ofanions and cations in the same test sample.

A water-containing eluant source 30 (preferably deionized water) isintroduced through a high pressure liquid chromatography (HPLC) pump 32.As those skilled in the art will appreciate, a variety of pumps may beused in this embodiment. However, a metal-free, reciprocating pistonpump is preferred, such as the ALLTECH Model 325 pump. The test sample,which contains anions to be detected, is injected through an injector34, and is routed by the eluant to column 36, which is preferablyconstructed as depicted in FIGS. 1-3. Again, as those skilled in the artwill appreciate, a variety of injectors may be used in the presentembodiment. However, metal-free, rotary 6-port injection valves arepreferred, such as those available from RHEODYNE (Model No. 9125) orVALCO. The column 36 comprises chromatography packing material. Foranion analysis, the column 36 is packed with an anion exchange packingmaterial (not shown). The anion exchange packing material preferablycomprises exchangeable hydroxide ions. By “exchangeable,” it is meantthat the hydroxide ions on the packing material may be displaced (orexchanged with) the sample anions. Suitable anion exchange packingmaterials comprise particles of primary, secondary, tertiary, orquaternary amino functionalities, either organic or inorganic. Preferredanion exchange packing materials comprise quaternary amino functionalityorganic or inorganic particles. These anion exchange particles may bepacked in the column in either resin form or impregnated in a membrane.Preferably, the anion exchange packing material is in resin form.

The sample anions and eluant are routed through column 36. In column 36,the sample anions displace the exchangeable hydroxide ions on the anionexchange packing material, and are retained in the column 36. Eitherjust before or after the sample anions are retained in column 36, anelectric current is generated in column 36 by turning on electricalpower supply 38. As those skilled in the art will appreciate, a varietyof electrical power supplies may suitably be used in the presentembodiment. All that is required for the electrical power supply is thatit be capable of providing from about 5-5,000 volts, more preferablyfrom about 28-2,800 volts, and most preferably about 10-1,000 volts tothe electrodes in the methods of electroelution chromatography describedherein. However, a time-programmable constant-current DC power supply ispreferred, such as the LABCONCO Model 3000 Electrophoresis Power Supply.Preferably, the cathode (not shown) of column 36 is located at anupstream end of the column 36, and the anode (not shown) is located at adownstream end of the column 36. Preferably, the electrodes arepositioned in the effluent flow channel (not shown) of the column 36.When the water in the eluant contacts the cathode (which is theelectrode located at the upstream end of the column 36 for anionanalysis), it undergoes electrolysis, and hydroxide ions are generatedaccording to the following reaction:Cathode: 2H₂O+2e ⁻→H₂(g)+2OH⁻Similarly, when the water in the eluant contacts the anode (which is theelectrode located at the downstream end of the column 36 for anionanalysis), it undergoes electrolysis, and hydronium ions are generatedaccording to the following reaction:Anode: 2H₂O→4H⁺+O₂(g)+2e ⁻

Thus, hydroxide ions and oxygen gas are generated at the upstream end ofthe column 36, and are routed through the column 36. The hydroxide ionsdisplace the retained sample anions on the anion exchange resin incolumn 36. The anion exchange resin is thus simultaneously regeneratedback to its hydroxide form while the sample anions are eluted. Thereleased sample anions and excess hydroxide ions generated at theupstream located cathode are routed to the downstream end of the column36 and combine with the hydronium ions generated at the downstreamlocated anode to form the highly conductive acid of the sample anionsand relatively non-conductive water, respectively. The effluent fromcolumn 36 (e.g., the column effluent), which contains the sample anionsin their highly conductive acid form and the relatively non-conductivewater, is then routed downstream to a detector 42 in which the sampleanions are detected The detector is preferably a conductivity detector,such as the ALLTECH Model 350 Conductivity Detector. The sample ionsdetected at the detector may also be quantified by a data system (notshown). The data system is preferably a computer based integrator, suchas the HEWLETT-PACKARD General Purpose Chemstation.

Preferably, the effluent from the detector (e.g., detector effluent) isthen routed from the detector 42 through a backpressure regulator 44.The backpressure regulator 44 keeps the gaseous H₂ and O₂ bubbles formedat the cathode and anode, respectively, small enough so that they do notinterfere with the detector 42. The backpressure regulator is preferablya spring-energized diaphragm system that maintains constant backpressureon the system regardless of flow rate, such as the ALLTECH backpressureregulator. The detector effluent can then be routed from thebackpressure regulator 44 to waste 46. Alternatively, before beingrouted to the detector 42, the column effluent may be routed through agas-permeable membrane tube (not shown) positioned between theanalytical column 36 and the detector 42. In that way, the gas bubblesgenerated by the electrolysis of water may be released through the gaspermeable membrane to the atmosphere.

In a preferred embodiment, the detector effluent is routed from theback-pressure regulator to an ion exchange bed 45. In anion analysis,the ion exchange bed may be packed with anion exchange packing material(not shown). The anion exchange packing material preferably comprisesexchangeable hydroxide ions, and is selected as previously describedwith respect to column 36. The sample anions replace the exchangeablehydroxide ions on the anion exchange packing material in the ionexchange bed, and the released hydroxide ions combine with the hydroniumcounterions of the sample anions to form water. The water may then berouted from the ion exchange bed 45 to the water-containing eluantsource 30. In this manner, a self-sustaining eluant source isestablished. Of course, the anion exchange resin in the ion exchange bed45 will eventually become exhausted, and thus it will need to beperiodically replaced or, in the alternative, regenerated according tothe methods described herein.

A variety of anions can be separated, detected, and analyzed accordingto the foregoing method. Examples include chloride ions, nitrate ions,bromide ions, nitrite ions, phosphate ions, sulfate ions, as well asother organic and inorganic anions. FIGS. 5A and 5B are schematic viewsof column 36 when used in the foregoing method of anion analysis byelectroelution chromatography. In FIG. 5A, the sample anions (X⁻) areretained on the anion exchange packing material 46 that is packed incolumn 36. Turning to FIG. 5B, when the power source (not shown) isturned on, hydroxide ions generated at the upstream located cathode ofthe column 36 are routed through the anion exchange packing material anddisplace the retained sample anions X⁻. The released sample anions (X⁻)combine with the hydronium ions generated at the downstream locatedanode of column 36 to form the highly conductive acid of the sampleanions (HX). Additionally, the excess hydroxide ions generated at theupstream located cathode combine with the hydronium ions generated atthe downstream located anode to form the relatively low conductivewater. Thereafter, the sample anions in their acid form are routed withwater from the column 36 to the detector (not shown) where the sampleanions are detected.

Based on the foregoing discussion, those skilled in the art willappreciate that the electrodes can be located either outside of orinside the column 36. The only necessary condition with respect to theplacement of the electrodes is that at least a portion of thechromatography packing material (anion exchange packing material in theforegoing embodiment) is disposed between the two electrodes, and thatfluid flow through the column is from one of the electrodes to theother. Thus, when it is said that the electrode is positioned at anupstream end of the column, it does not necessarily mean that theelectrode is actually located in the column. To the contrary, it issimply meant that the electrode is located between the fluid source andthe other electrode. Similarly, by the term “downstream end” of thecolumn, it is meant that the electrode is located on the side oppositethe fluid source relative to the other electrode. Again, the electrodeis not necessarily positioned in the column itself. Thus, fluid flow isalways from the “upstream” located electrode to the “downstream” locatedelectrode.

Without being restricted to theory, it is presently believed that theelectrical current in column 36 is generated between the two electrodesvia ion transport along the chromatography packing material (not shown)in the column 36. However, where the packing material is not capable ofion transport, it is presently believed that ion transport takes placevia the mobile phase. This electric current via ion transportsurprisingly occurs even when the chromatography packing material andthe eluant may not inherently be electrically conductive. Because theelectric current is generated by ion transport along the chromatographypacking material in the effluent flow channel (not shown) of column 36,the electric current through column 36 is in the same direction as fluidflow through the column 36.

As those skilled in the art will understand, the electrical voltagegenerated in column 36 must be of sufficient strength for theelectrolysis of water to occur. The strength of the current generated incolumn 36 is directly proportional to the voltage applied at theelectrodes, the cross-section area of the electrodes, and the capacityof the packing material in column 36 (e.g. the higher the capacity ofthe packing material, the lower the resistance is in the column 36). Thestrength of the current in column 36 is inversely related to thedistance between the two electrodes.

The above method and apparatus can also be adapted for the separation,detection, and analysis of sample cations as well. For cation analysis,the column 36 is packed with cation exchange packing material. Thecation exchange packing material preferably comprises exchangeablehydronium ions. Preferred cation exchange packing materials include acidfunctionalized organic and inorganic particles, such as phosphoric acidfunctionalized organic or inorganic particles, carboxylic acidfunctionalized organic or inorganic particles, sulfonic acidfunctionalized organic or inorganic particles, and phenolic acidfunctionalized organic or inorganic particles. The cation exchangeparticles may be packed into the column in either resin form orimpregnated into a membrane. The most preferred cation exchange packingmaterials are sulfonic acid functionalized particles. Most preferably,the cation exchange packing material is packed in the column in resinform.

In cation analysis, the apparatus of FIG. 4 is further reconfigured sothat the anode (not shown) is positioned at the upstream end of thecolumn 36 and the cathode (not shown) is positioned at the downstreamend of the column 36. Thus, when an electric current of sufficientstrength is applied, hydronium ions are generated at the upstreamlocated anode 36. The hydronium ions are then routed across the cationexchange packing material and displace the previously retained samplecations in the column 36. The released sample cations and excesshydronium ions generated at the upstream located anode combine with thehydroxide ions generated at the downstream located cathode to form thehighly conductive bases of the sample cations and water, respectively.The sample cations, in their basic form, and relatively non-conductivewater are then routed to detector 42 where the sample cations aredetected. Finally, the detector effluent is preferably routed throughion exchange bed 45, which comprises cation exchange packing material(not shown). The cation exchange packing material preferably comprisesexchangeable hydronium ions, and is selected as described above. Thesample cations displace the hydronium ions and are retained in the ionexchange bed 45, and the released hydronium ions combine with thehydroxide counterions of the sample cations to form water. The ionexchange bed effluent (which comprises water), is then routed to thewater containing eluant source.

With reference back to FIG. 4, in an especially preferred embodiment ofthe present invention both cation exchange packing material (not shown)and anion exchange packing material (not shown) is packed in column 36.Similarly, ion exchange bed 45 is packed with both cation exchangepacking material (not shown) and anion exchange packing material (notshown). Preferably, the cation and anion exchange packing materialcomprise exchangeable hydronium and hydroxide ions, respectively, andare selected as previously described. In this embodiment, both cationsand anions in the same test sample may be analyzed according to theforegoing method of electroelution chromatography. However, thisconfiguration is also preferred even when only cations or only anionsare being detected as well.

However, when it is desired to separate both anions and cations in thesame sample, a sample comprising anions and cations to be separated isrouted through column 36. The sample cations are retained in column 36on the cation exchange resin and the sample anions are retained incolumn 36 on the anion exchange resin. The polarity of column 36 isarranged depending on whether cations or anions are to be eluted first.Where it is desired to elute the cations first, the anode (not shown) islocated at an upstream end of column 36 and the cathode (not shown) islocated at a downstream end of the column 36. The power source 38 isturned on to generate an electric current across the anion exchangeresin and cation exchange resin in column 36. Hydronium ions aregenerated at the upstream located anode by the electrolysis of thewater-containing eluant as previously described. Similarly, hydroxideions are generated at the downstream located cathode as previouslydescribed. The hydronium ions are routed through column 36, displacingthe retained sample cations and simultaneously regenerating the cationexchange packing material back to its hydronium form. The releasedsample cations and the excess hydronium ions generated at the upstreamlocated anode combine with hydroxide ions generated at the downstreamlocated cathode to form the highly conductive bases of the samplecations and the relatively non-conductive water, respectively.

The sample cations (in their base form) and water are then routed todetector 42 where the sample cations are detected. The detector effluentmay then be routed to the ion exchange bed 45 where the sample cationsare retained by displacing the hydronium ions on the cation exchangepacking material in the ion exchange bed 45. The hydronium ionsdisplaced from the cation exchange packing material combine with thehydroxide counterions of the sample cations to form water, which maythen be routed to the water-containing eluant source 30.

After the sample cations have been detected, the polarity of column 36is reversed, so that the cathode (not shown) is located at an upstreamend of column 36 and the anode (not shown) is located at a downstreamend of the column 36. Hydroxide ions are generated at the upstreamlocated cathode and hydronium ions are generated at the downstreamlocated anode by electrolysis of the water-containing eluant aspreviously described. The hydroxide ions are routed through column 36,displacing the retained sample anions and simultaneously regeneratingthe anion exchange packing material back to its hydroxide form. Thereleased sample anions and the excess hydroxide ions generated at theupstream located cathode combine with the hydronium ions generated atthe downstream located anode to form the highly conductive acids of thesample anions and the relatively non-conductive water, respectively.

The sample anions (in their acid form) and water are routed to thedetector 42 where the sample anions are detected. The detector effluentmay then be routed to the ion exchange bed 45 where the sample anionsare retained by displacing the hydroxide ions on the anion exchangepacking material in the ion exchange bed 45. The displaced hydroxideions then combine with the hydronium counterions of the sample anion toform water, which may then be routed to the water-containing eluantsource 30.

In another embodiment of the present invention, two columns asillustrated in FIG. 1 can be arranged in series for use in methods ofdetecting cations and anions in the same test sample. With reference toFIG. 6, a water-containing eluant source 30 (again, preferably deionizedwater) is introduced through a high pressure liquid chromatography(HPLC) pump 32. A test sample containing both anions and cations to bedetected is injected through an injector 34, and is carried by theeluant to a first column 36 of the present invention, which is packedwith anion exchange packing material (not shown). The anion exchangepacking material preferably comprises exchangeable hydroxide ions, andis selected as previously described.

The sample anions displace the hydroxide ions on the anion exchangeresin and are retained in the first column 36. The first columneffluent, which contains the sample cations and displaced hydroxideions, is routed to a second column 136. Column 136 is packed with cationexchange packing material preferably comprising exchangeable hydroniumions, and is selected as previously described. The sample cationsdisplace the hydronium ions on the cation exchange packing material andare retained in the column 136. The released hydronium ions neutralizethe hydroxide ions in the first column effluent to form water. The wateris then preferably routed through the detector 42 (giving no signal),through an ion exchange bed 46, through valve 48 and back to the eluantsource 30.

An electric current sufficient to electrolyze water is then generated incolumn 36 across the anion exchange packing material by turning onelectric power supply 38. The cathode (not shown) of the column 36 islocated at an upstream end and the anode (not shown) is located at adownstream end of the column 36. The water-containing eluant undergoeselectrolysis at the upstream located cathode thereby generatinghydroxide ions and hydrogen gas as previously described. The hydroxideions generated at the upstream located cathode are routed through thecolumn 36 and displace the retained sample anions on the anion exchangepacking material thereby eluting the sample anions from column 36.

At the downstream located anode of the column 36, hydronium ions andoxygen gas are generated as previously described by the electrolysis ofthe water-containing eluant. The released sample anions from column 36and the excess hydroxide ions generated at the upstream located anode ofthe column 36 combine with the hydronium ions generated at thedownstream located cathode of the column 36, to form the highlyconductive acids of the sample anions and relatively non-conductivewater, respectively.

The acids of the sample anions and water from column 36 are routedthrough column 136 unretained to detector 42, in which the sample anionsare detected and quantified by a data system 43. The bubbles formed bythe hydrogen gas and oxygen gas generated at the cathode and anode,respectively, of the column 36 are kept small enough by back pressureregulator 44 so that they do not interfere with the detector 42.

The detector effluent is then preferably routed from the detector 42through back pressure regulator 44 to an ion exchange bed 46 comprisingion exchange packing material having exchangeable hydronium andexchangeable hydroxide ions. The ion exchange bed is preferably ofhigh-purity Such as those used to produce deionized water. The sampleanions displace the hydroxide ions and are retained in the ion exchangebed 46. The displaced hydroxide ions neutralize the resulting hydroniumcounterions of the sample anions to form water, which is preferablyrouted through valve 48 back to the eluent source 30.

Once the sample anions have been detected, an electric currentsufficient to electrolyze water is then generated in column 136 acrossthe cation exchange packing material by turning on electric power source138. The anode (not shown) is positioned at an upstream end of thecolumn 136 and the cathode (not shown) is located at a downstream end ofthe column 136. The water containing eluant undergoes electrolysis atthe upstream located anode of the column 136 thereby generatinghydronium ions. The hydronium ions are then routed through the column136 and displace the previously retained sample cations on the cationexchange resin thereby eluting the sample cations from column 136.

At the downstream located cathode (not shown) of column 136, hydroxideions and hydrogen gas are generated as previously described by theelectrolysis of the water-containing eluant. The released sample cationsand the excess hydronium ions generated at the upstream located cathodeof the column 136 combine with the hydroxide ions generated at thedownstream located anode of the column 136, to form the highlyconductive bases of the sample cations and relatively non-conductivewater, respectively.

The sample cations (in their base form) and water are then routed fromcolumn 136 to detector 42, in which the sample cations are detected andquantified by data system 43. Again, the bubbles formed by the hydrogengas and oxygen gas generated at the cathode and anode, respectively, ofthe column 136 are kept small enough by the backpressure regulator 44 sothat they do not interfere with the detector 42.

The detector effluent is then preferably routed from the detector 42through backpressure regulator 44 to the ion exchange bed 46. The samplecations displace the exchangeable hydronium ions in the ion exchange bed46 and are retained therein. The displaced hydronium ions neutralize thehydroxide counterions of the sample cations to form water, which ispreferably routed through valve 48 back to eluant source 30.

FIG. 7 is a chromatogram of a test sample containing both anions andcations separated pursuant to the foregoing method and apparatus of thepresent invention. The first series of peaks represent the sample anionsthat are eluted when power is applied and an electric current isgenerated across the anion exchange packing material in column 36. Thesecond series of peaks represent the sample cations that are eluted anddetected when power is applied and an electric current is generatedacross the cation exchange packing material in column 136.

As those skilled in the art will recognize, the foregoing apparatus andmethod can be easily reconfigured so that the column 36 is packed withcation exchange packing material and the anode is located at an upstreamend and the cathode is located at a downstream end of the column 36, andcolumn 136 is packed with anion exchange packing material and thecathode is located at a upstream end and the anode is located at adownstream end of the column 136.

The foregoing methods and apparatuses provide many advantages. Forexample, the strength of the current applied in the columns 36 and 136will determine the concentration of hydroxide and hydronium ionsgenerated in these columns. The higher the current, the greater theconcentration of hydroxide or hydronium ions and the easier and quickerthe sample anions and cations will be eluted. Thus, gradients aretherefore possible through time-based current programming in the anionand cation columns. Moreover, the foregoing method can be configured asa closed loop system. Additionally, simultaneous cation and anionanalysis is possible with high sensitivity and low background noise.Moreover, water dips, and unretained counter-cation peaks, andunretained counter-anion peaks often present in traditional ionchromatography methods may be reduced and even eliminated.

The methods and apparatuses previously described can also be used inmethods and apparatuses for the electroelution of proteins, as well asany other test sample whose affinity for chromatographic packingmaterial is affected by Ph changes and/or ionic strength changes.Proteins and many other test samples retain on affinity stationaryphases by biological recognition, on reversed phases by hydrophobicinteractions, on ion-exchange or chelation packing materials by chargeinteractions, by size on size exclusion packing materials, byhydrophobic interactions on hydrophobic packing materials, and by normalphase interactions on normal phase packing materials. All of theseretention mechanisms may be mediated by ionic strength and/or Ph. Byusing the electrolysis of water as discussed in the foregoingembodiment, the hydrogen and hydroxide ion concentration in the columnillustrated in FIG. 1 can be controlled, thereby permitting the controlof the Ph and ionic strength inside the column. Thus, by packing thechromatography column of the present invention with an ion-exchange,affinity stationary phase, reversed phase, size exclusion, chelating,hydrophobic, or normal phase chromatography packing material, proteinsor other samples can be retained when no power is applied to the column.However, when power is applied to generate hydronium ions (decrease Ph)or hydroxide ions (increase Ph) within the column, the resulting ionicstrength and Ph change may be used to elute the retained protein (orother samples) from the column. Thus, the column of the presentinvention can be used to separate and purify a wide range of compoundson both an analytical and preparative scale.

Suitable packing materials for use in the foregoing embodiment includeProtein A affinity packing material as the affinity phase packingmaterial; C-18 reversed phase packing material as the reversed phasepacking material; Chelex-100 from Bio-Rad in the chelating packingmaterial; ALLTECH Macrosphere GPC as the size exclusion packingmaterial; SYNCHROM SynChropak HIC as the hydrophobic packing material;and ALLTECH Alltima Silica as the normal phase packing material.

Additionally, as discussed below, the column of the present inventioncan also be used as a self-regenerating solid phase chemical suppressorin various apparatuses and chromatography methods.

B. Electrochemically Regenerated

Solid Phase Chemical Suppressor

1. System Configurations for Anion Analysis

FIGS. 8A-8D are schematic views of various preferred apparatusconfigurations for a preferred method of ion analysis using the columnillustrated in FIG. 1 as a self-regenerating solid phase chemicalsuppressor. With reference to FIG. 8A, an eluant source 202 is in fluidconnection with a pump 204. Downstream from the pump 204 is an injector206 where a test sample can be added to the system Located downstreamfrom the injector 206 is an analytical (or chromatography) column 208where separation of the ions in the test sample occurs. In anionanalysis, a low-capacity anion exchange column is preferably used. Forcation analysis, a low-capacity cation exchange column is preferablyused.

Located downstream from, and in fluid connection with, the analyticalcolumn 208, is a 10-port switching valve 210 The switching valve ispreferably of the metal-free rotary type. In connection with the 10-portswitching valve 210 are two columns, 212 and 214, respectively, asillustrated in FIG. 1. Connected to columns 212 and 214 are electricalpower sources 216 a and 216 b, respectively. One power source connectedto both columns 212 and 214 may be used as well. This embodimentgenerally requires lower voltage than the foregoing methods ofelectroelution chromatography because the capacity of the chromatographypacking materials in this embodiment is greater (e.g. lower resistance),and thus lower voltages are capable of generating a current sufficientfor the electrolysis of water. A preferred power source is the KENWOODPR 36-1.2 power supply. When the column illustrated in FIG. 1 is used asa suppressor, the power source should be capable of delivering about1-100 volts to the electrodes, more preferably about 10-90 volts, andmost preferably about 3-15 volts. Finally, a conductivity detector 218is connected with 10 port switching valve 210. As described in moredetail below, the column illustrated in FIG. 1 is adapted for use as aself-regenerating solid phase chemical suppressor in the foregoingconfiguration.

Still with reference to FIG. 8A, an aqueous eluant source 202 introduceseluant through a HPLC pump 204. A test sample containing anions to bedetected is injected through injector 206, and is routed by the eluantto analytical (or chromatography) column 208. In the present embodiment(e.g., anion analysis) the eluant may comprise solutions of sodiumcarbonate, sodium bicarbonate, sodium hydroxide or some other base thatis converted to a weak acid by counterion exchange with hydronium ions.The most preferred eluant for anion analysis are solutions of sodiumhydroxide.

The analytical column 208 is preferably packed with anion exchangepacking material (not shown). Suitable anion exchange packing materialscomprise particles of primary, secondary, tertiary, or quaternary aminofunctionalities, either organic or inorganic. The preferred anionexchange packing materials comprise quaternary amino functionalityorganic or inorganic particles. These anion exchange particles mayeither be packed into the column in resin form or impregnated into amembrane. Preferably, the packing material is in resin form.

Different anions in the test sample have differing affinities for theanion exchange packing material in the analytical column 208. Thestronger the affinity of a particular type of anion for the packingmaterial in the analytical column 208, the longer that type of anionwill be retained in the column 208. Conversely, the weaker the affinityof a particular type of anion for the packing material in the analyticalcolumn 208, the shorter that particular type of anion will be retainedin the column 208. Thus, because different anions have differentaffinities for the packing material in column 208, the sample anions areeluted at different speeds from the column 208 and are thereforeseparated or resolved.

The effluent from analytical column 208 (hereinafter referred to as“chromatography effluent”) is routed from the column 208 through 10-portswitching valve 210 to column 212. In this embodiment, the column 212 isadapted for use as a suppressor in a method of anion analysis. Thecolumn 212 is packed with cation exchange packing material (not shown).Preferred cation exchange packing materials include acid functionalizedorganic or inorganic particles, such as phosphoric acid functionalizedorganic or inorganic particles, carboxylic acid functionalized organicor inorganic particles, phenolic acid functionalized organic orinorganic particles, and sulfonic acid functionalized inorganic ororganic particles. The cation exchange particles may either be packedinto the column in resin form or impregnated into a membrane. The mostpreferred cation exchange packing materials are sulfonic acidfunctionalized inorganic or organic particles in resin form.

Two ion-exchange reactions take place in the suppressor 212:

1. Eluant:

-   -   (where the eluant is sodium hydroxide and the cation exchange        packing material comprises sulfonic acid functionalized        particles):        NaOH+Resin−SO₃ ⁻H⁺→Resin−SO₃ ⁻Na⁺+H₂O

2. Analyte:NaX+Resin−SO₃ ⁻H⁺→Resin−SO₃ ⁻Na⁺+HX

-   -   (where X=anions such as Cl, NO₂, Br etc.)        The sodium ions in the high conductivity eluant are removed by        ion exchange with the hydronium ions present on the cation        exchange packing material in the column 212. The high        conductivity sodium hydroxide eluant is thus converted to the        relatively non-conductive water (the sample counterions are also        suppressed by ion exchange with hydronium ions on the cation        exchange packing material). This, of course, reduces the        background noise from the eluant (and sample counterions) when        the sample anions are ultimately detected in the detector 218.        The sample anions are converted into their highly conductive        acid form by exchanging their counterions with hydronium ions on        the cation exchange packing material in the column 212. As can        be ascertained from the above reactions, the eluant in anion        analysis can be any salt solution that forms a weakly conductive        acid in the suppressor 212. Examples of suitable eluants include        aqueous solutions of sodium hydroxide, sodium        carbonate/bicarbonate, and sodium tetraborate. The eluant must        further comprise water, however, to feed the electrolysis in the        methods of the invention.

After the eluant has been converted to its weak acid and the sampleanions to their highly conductive acids in the column 212, thesuppressor effluent is routed through the 10-port switching valve 210 todetector 218 where the sample anions are detected. Data from thedetection of the sample anions is preferably recorded on a chart, graph,an integrator, a computer, or other recording means (not shown). Theeffluent from the detector 218 (hereinafter referred to as “detectoreffluent”) is then routed through 10-port switching valve 210, andcolumn 214 to waste.

When the cation exchange packing material in suppressor 212 is exhausted(e.g., completely converted from the hydrogen to sodium form), a sharpincrease in the conductance of the suppressor effluent is observed.Before this happens, the 10-port switching valve 210 is switched to theconfiguration depicted in FIG. 8B. While using the column 214 tosuppress the chromatography effluent in the same manner as previouslydescribed with respect to column 212, the detector effluent is recycledback through 10-port switching valve 210 to the exhausted suppressor 212to regenerate it as follows.

A power source 216 a is turned on thereby generating an electric currentsufficient for the electrolysis of water across the exhausted cationexchange packing material in suppressor 212. Suppressor 212 isconfigured so that the anode (not shown) is positioned at the upstreamend and the cathode (not shown) is positioned at the downstream end ofthe suppressor 212. The detector effluent (which contains water)undergoes electrolysis at the upstream located anode of suppressor 212as previously described:2H₂O→4H⁺+O₂+4e ⁻Hydronium ions and oxygen gas are thus generated at the upstream anodeof suppressor 212. Since the detector effluent is flowing from the anodeside to the cathode side of the suppressor 212, the hydronium ions arerouted across the exhausted cation exchange packing material insuppressor 212 converting it back to the hydrogen form according to thefollowing reaction:Resin−SO₃ ⁻Na⁺+H⁺→Resin−SO₃ ⁻H⁺+Na⁺The oxygen gas and displaced sodium ions (and sample counterions) fromsuppressor 212 are then routed through 10-port switching valve 210 towaste.

At the downstream located cathode of suppressor 212, water undergoeselectrolysis as previously described:2H₂O+2e ⁻→H₂+2OH⁻Hydroxide ions and hydrogen gas are thus generated at the downstream endof suppressor 212. The hydroxide ions and hydrogen gas generated insuppressor 212 are routed through 10-port switching valve 10 to waste.

Alternately, the systems waste products may be recycled. As can beappreciated from the above chemical reactions, the regeneration processliberates exactly the same mass of eluant ions consumed during thesuppression process. Because the process is quantitative, routing thewaste products from switching valve 10 back to eluant source 2 on acontinuous basis will result in continuous reconstitution of theoriginal eluant (in this embodiment sodium hydroxide), eliminatingchemical waste.

Power source 216 a is left on long enough to regenerate the cationexchange packing material in suppressor 212. Once the suppressor 212 isregenerated, the power source is turned off. The detector effluent isstill preferably routed through the suppressor 212, however, for a timesufficient to purge any remaining gas bubbles and electrolysis productsin suppressor 212, and to equilibrate the column. Once the suppressor212 is regenerated and equilibrated, it is ready for use as the “active”suppressor when suppressor 214 becomes exhausted. Once suppressor 212 isready to go back on line, the analytical column effluent is re-routed tosuppressor 212 and suppressor 214 is regenerated in the same manner aspreviously described with respect to suppressor 212.

The foregoing arrangement allows endless cycling between the twosuppressors 212 and 214 for continuous instrument operation withoutinterruption or suppressor replacement. Preferably, when the eluantcomprises an organic such as methanol, after regeneration the eluant isallowed to pass through the suppressor for a time (usually about 5minutes) sufficient to wash unwanted components that may remain in thesuppressor after regeneration. Once these unwanted by-products arepurged from the suppressor, the sample to be analyzed may then beinjected into the system. The system is easily automated using automaticvalves and power supplies to provide unattended operation for extendedperiods.

In one aspect of the invention, a computer program is used to implementand automate the switching between suppressors. One presently preferredflowchart providing an overview of such computer program and itsfunctions is provided in FIGS. 23( a)-(c). A computer program followingthe procedures and functions shown in FIGS. 23( a)-(c) is provided inthe Appendix. The presently preferred computer program is written in theForth computer language although, as those skilled in the art willappreciate, the flowchart shown in FIGS. 23( a)-(c) can be implementedin any computer language without departing from the spirit and scope ofthe invention.

In a presently preferred embodiment, the above-referenced software willbe used to run a self-contained suppressor system comprising twosolid-phase electrochemical suppressors packed with ion exchange resin,a two position electrically actuated 10-port switching valve, a constantcurrent power supply and a microprocessor for executing the computersoftware attached in Appendix A. These components are preferably housedtogether in one housing. A suitable microprocessor is the MOTOROLA 8-bitmicroprocessor (Motorola Part No. 68HCP11 A1 FN) operating with a 4 mHzcrystal, 32 K×8 EPROM, National Part No. 27C256 and 8K×8 RAM, MOSELModel Part No. MS 6264C-80PC.

With respect to this suppressor system, the mobile phase from theseparator (or analytical) column flows through one suppressor at a time.While one suppressor is being used, the other is electrochemicallyregenerated and equilibrated. The valve switches between suppressorsafter each sample injection, providing a fresh suppressor cell for eachanalysis. Preferably, the suppressors will be of relatively small volumeto avoid problems such as band broadening, Donnan exclusion, and theoxidation of nitrite to nitrate associated with conventional packed-bedsuppressors. The suppressors are preferably either 7.0 by 7.5 mm or14×7.5 mm in internal diameter. Finally, the operator interface on thefront panel of the suppressor unit has a series of buttons or keys(which are discussed below) for easy operation of the unit by labpersonnel.

The ion exchange resin in the suppressors is preferably visible when thesuppressors are housed in the suppressor unit so that suppressor statusmay be monitored at all times. This is accomplished by housing thesuppressors in a compartment having a transparent cover on a front panelor operator interface of the unit. The suppressor status may beindicated by coating the ion-exchange resin with an inert dye, whichchanges color as the suppressor resin becomes exhausted. For anionanalysis, the suppressors are packed with cation exchange resin in thehydrogen form, and the resin is coated with an inert dye comprisingquinaldine red. The unexhausted resin thus has a gold color, whichchanges to magenta during suppression as the hydrogen ions on the resinare replaced by the mobile phase and sample counterions. For cationanalysis, the suppressors are packed with anion exchange resin in thehydroxide form, and the resin is coated with an inert dye comprisingthymolphthalein. The unexhausted resin thus has a blue color, whichchanges to beige during suppression as the hydroxide ions on the resinare replaced by the mobile phase and sample counterions.

A brochure for a self-contained suppressor unit according to onepreferred aspect of the invention is attached in the Appendix. Thisbrochure (e.g. Alltech, Bulletin #334) refers to Alltech's ERIS™ 1000Autosuppressor, and is incorporated herein by reference. The softwarepresently contemplated for use with the Alltech ERIS™ 1000Autosuppressor is attached in the Appendix and a flowchart providing anoverview of this software is at FIGS. 23( a)-(c). Among other things,the software automatically coordinates suppression and regenerationbased on data inputted by the operator and based on signals receivedfrom other external devices in the chromatography system. The softwarealso generates messages on a system display concerning operationalparameters, regeneration parameters and status and system errors. Thesystem display is preferably a four line alpha numeric display and ispositioned on the operator interface of the suppressor unit. A copy ofthe most current draft of the Operator's Manual for the Alltech ERIS™1000 Autosuppressor is attached in the Appendix. This Operator's Manualis also incorporated herein by reference.

With reference to FIGS. 23( a)-(c), Step 500 is the beginning of thesoftware and depicts the startup screen when the suppressor unit ispowered-up. Program flow then proceeds to step 501. At step 501, amessage is generated on the system display indicating the “Active”method, which is the method presently inputted in the system for thenext sample injection. The “Active ID” has a one or two digit numberhaving associated pre-assigned parameter values for a given method. Thesystem may store up to twelve different “Active ID” numbers, each havingpre-assigned method parameters. The system comes with two preassigned“Active ID” “numbers,” EPA-A and EPA-B (EPA Method 300, parts A and B,respectively). If selected, “EPA-A” or “EPA-B” will appear on the systemdisplay as the “Active ID”. Additionally, ten “Active ID” numbers (e.g.1-10) may be inputted into the system wherein each number has its ownunique operating parameters. In addition to calling up any one of thetwelve Active ID numbers having pre-assigned parameters, new operatingparameters may also be inputted into the system for a particular samplerun by depressing the “Select Method” button on the operator interface.The procedure for inputting new operating parameters will be discussedin more detail below.

Referring back to the message at step 501, the three digit numberunderneath the “Type” heading provides information concerning the typeand capacity of the individual suppressors. In step 501, the threenumbers corresponding to the “type” heading are “312.” The first digit(e.g., “3”) refers to the type of sample analysis (i.e., either anion orcation analysis), and, thus, the type of suppressor in the system. Thesystem will automatically set the polarity of the electrodes based onthis entry. For example, the number “3” signifies anion analysis. Thistells the system to configure the electrode positioned at the detectoreffluent end of the suppressor as the anode. Conversely, if the number“2” were entered in place of “3” the system would automatically reversethe polarity of the electrodes making the electrode positioned at thedetector effluent end of the suppressor the cathode. The next two digitsunder the “Type” heading (e.g., “12”) refer to the capacity of theindividual suppressors in milliequivalents (meq.) times 10. Thus, thescreen at step 501 indicates a suppressor for anion analysis having acapacity of 1.2 milliequivalents. In a preferred embodiment, thesuppressors themselves will have a label or some other attached meansfor displaying a three digit number to correspond to the “Type” numberdiscussed above. Thus, for example, a suppressor bearing the number“312” is for anion analysis and has a capacity of 1.2 milliequilvalents.As those skilled in the art will appreciate, the two suppressors shouldhave the same capacity in this embodiment.

Still with respect to the message generated at step 501, the “Flow”heading refers to the flowrate in mL/min. for the analysis. The screenat step 501 indicates a flowrate of 1.0 mL/min. The “Conc.” heading inthe message generated at step 501 refers to the concentration of themobile phase in milliequivalents/Liter (meq./L). For anion analysis, themeq./L of cations in the mobile phase is indicated Conversely, forcation analysis, the meq./L of anions in the mobile phase is indicated.Because the message at step 501 indicates anion analysis, theconcentration of 4.0 meq./L refers to the concentration of cations inthe mobile phase. Finally, the “Time” heading in the message generatedat step 501, refers to the total run time for the analysis to a tenth ofa minute.

To exit the screen at step 501 and proceed to the next screen theoperator merely depresses an “Enter” button on the operator interfaceand program flow will proceed to step 502. In step 502 a message isgenerated on the system display asking whether the operator wishes toproceed with the method having the parameters indicated at step 501. Ifyes, the operator will then depress a “Next/Yes” button and then the“Enter” button on the operator interface and program flow proceeds tostep 503. If the answer is “No,” then the operator may depress a“Back/No” button and then the “Enter” button on the operator interfaceto recall the screen at step 501. The operator may then edit theparameters indicated at step 501 by depressing a “Select Method” buttonkey on the operator interface. Details concerning inputting new methodparameters are discussed in further detail below.

Referring back to step 503, a message is generated on the system displayrequesting that the suppressor (“Cell”) color be checked. If the colorcondition is “O.K.,” that is, if the color indicates that thesuppressors are in a condition to accept a sample injection, theoperator may depress the “Next/Yes” and then the “Enter” buttons on theoperator interface and the program flow proceeds to step 504. The systemis now ready to receive a sample injection.

The system is programmed to switch between suppressors after each sampleinjection. Samples may be injected manually or automatically. For manualinjection, an “Inject/Start” button is provided on the user interface,which should be depressed simultaneously with the introduction of thesample. Additionally, pins located on the rear panel of the operatorinterface are designed to accept a sample injection signal from anexternal device such as an autosampler or a manual injection valveequipped with a position sensing switch. While waiting for a sampleinjection, the system will cycle the mobile phase between the twosuppressor cells. Once the total analysis time elapses for theparticular method inputted into the system, the system willautomatically direct the mobile phase to a fresh suppressor and willbegin counting down the total analysis time. Upon receiving an injectionsignal, the system resets the total analysis time, but continuesdirecting column effluent to the active suppressor because the newsuppressor has not yet “seen” an injection) The valve will automaticallyrotate to the other suppressor the next time the system receives aninjection signal or when the analysis time has elapsed, whichever comesfirst. The system software thus ensures that only one sample injectionis allowed to flow through the suppressor between regeneration cyclesregardless of when the sample is injected.

Referring back to step 504, a message is generated on the system displaypertaining to the “Method ID”. The Method ID indicates the currentoperating parameters (e.g., cell type, flowrate, eluant concentrationand analysis run time), and the time remaining until the sample analysisis complete. While one suppressor is in the suppressing mode the othersuppressor is regenerated by the electrolysis of the detector effluent.By depressing the “Next/Yes” button on the operator interface, a messageis generated at step 505. This screen shows the total regeneration (orflushing) time in minutes and seconds (min.:sec), the remainingregeneration time (or flushing) time (min.:sec.), how much current isapplied to the electrodes in the suppressor being regenerated, and thevoltage across the suppressor being regenerated.

Based on the flowrate, mobile phase concentration and total run timeentered for the particular method, the system automatically calculatesthe amount of current required to completely regenerate the suppressorwithin 40% of the run time. A negative voltage indicates that thesuppressor being regenerated is for anion analysis. A positive voltageindicates that the cell being regenerated is for cation analysis. Themessage at step 505 indicates that the left suppressor is beingregenerated while the right suppressor is in the active or suppressingmode. The operator may toggle between the screens displayed in steps 504and 505, respectively, by depressing the “Next/Yes” and “Back/No”buttons on the operator interface. The current is applied to thesuppressor undergoing regeneration just long enough to regenerate thesuppressor (as calculated by the system based on the method parameters)and then the current is automatically shut off when regeneration iscomplete. However, detector effluent continues to flow through theregenerated suppressor to purge any remaining gas bubbles and otherelectrolysis by-products.

The operator interface also has a cell (e.g., suppressor) statusdisplay. This display indicates the cell is “IN USE”, and whether theother cell is either being “REGEN” (e.g., regenerated) or is “READY”(i.e., regeneration has been completed). The suppressors are positionedin a compartment on the operator interface and the compartment has atransparent door so that the suppressors are visible to the operator.The cell status display is positioned above the compartment housing thesuppressors and comprises a separate display for each of the “LEFT” and“RIGHT” suppressors, respectively. Thus, for example, if the LEFTsuppressor is on line (i.e., in the active or suppressing mode), thecell status display for the “LEFT” suppressor will display an “IN USE”message. While the LEFT suppressor is in use, the cell display for the“RIGHT” suppressor will display either a “REGEN” or a “READY” message,depending on whether the “RIGHT” suppressor is being regenerated orregeneration has been completed, respectively.

If for any reason the operator desires to regenerate both of thesuppressors in the system, the operator may depress a “FULL REGEN” (e.g.Full Regeneration) button on the operator interface. Program flow thenproceeds to step 511. The “FULL REGEN” button may be depressed atanytime during the display of the “Method ID” screen. A message isgenerated on the system display at step 511 asking for confirmation thatsuppressor regeneration is desired. By depressing the “Next/Yes” buttonon the operator interface, program flow then proceeds to step 512 wherea message is generated on the system display requesting the operator to“Check Cell Condition”. If the color of the resin in the suppressorsindicates that the suppressors are in the unexhausted form, the operatormay depress the “Next/Yes” and then the “Enter” buttons on the operatorinterface and program flow proceeds to step 504. In which case, thesystem is ready to receive a sample injection. Conversely, if the resincolor indicates that the suppressors are exhausted, and thusregeneration is desirable, the operator may depress the “Back/No” andthen the “Enter” buttons on the operator interface and program flowproceeds to step 513.

The system then begins to regenerate both of the suppressors, and amessage is generated on the system display at step 513 giving the totaltime (min.:sec.) required to regenerate both suppressors and theremaining time (min.:sec.) until both suppressors are regenerated.Again, the time to regenerate the suppressors is calculated by thesystem based on the operating or method parameters inputted by theoperator. Only one suppressor is regenerated at a time, and specificinformation concerning the suppressor being regenerated may be obtainedby depressing the “Next/Yes” button on the operator interface at Step513 and program flow will proceed to step 514. At step 514 a messagesimilar to that generated at step 505 is generated on the systemdisplay. This message reports on the status of the suppressor undergoingregeneration at that moment, which in step 514 is the left suppressor.The operator may toggle between screens 513 and 514 by depressing the“Next/Yes” and “Back/No” buttons on the operator interface. Once boththe suppressors are regenerated, program flow then proceeds to step 504and the system is ready to accept a sample injection. Also, the programallows for aborting regeneration of the suppressors at any time bysimply depressing the “Enter” button on the operator interface at step513. This will abort the regeneration sequence and program flow willproceed to step 503.

Method parameters may be entered or changed at the beginning of thesoftware, or at any other time by simply depressing the “Select Method”(Step 520) button on the operator interface. Program flow then proceedsto step 521. At step 521, a message is generated on the system displayindicating the method parameters. By depressing the “Next/Yes” and“Back/No” buttons on the operator interface the operator may scrollthrough the parameters—Type, Flow, Conc. and Time. Arrow buttons on theoperator interface are used to increase or decrease the numerical valuesfor each of these parameters. Once the desired operating parameters areset, the “Enter” button on the operator interface is depressed andprogram flow then proceeds to step 522. A message is generated on thesystem display at step 522. If the operator wishes to proceed with themethod parameters selected at step 521, the “Next/Yes” and then the“Enter” buttons are depressed at step 522 and program flow proceeds tostep 504 and the system is ready to accept a sample injection.Conversely, if the operator does not wish to proceed with the parametersselected in step 521, the “Back/No” and then the “Enter” buttons aredepressed on the operator interface at step 522 and program flowproceeds back to step 521.

When selecting method parameters at step 521, certain considerationsmust be taken into account. When entering the three digit number for“Type” of method, the type of analysis (i.e., whether cation or anionanalysis) and the capacity (in meq./L) of the suppressors in the systemmust be entered. When entering the flow rate (e.g., “Flow”), theoperator must match the flow rate with that of the HPLC pump in thechromatography system. When entering the concentration of the mobilephase (“Conc.”), the concentration (in meq./L) of the mobile phasecounter-ions of the sample ions must be entered. Exemplary calculationsand a list of meq/L. values for common mobile phases are provided inAppendix C to the Operator's Manual in the Appendix. Finally, the totalrun time (“Time”) for the analysis to the tenth of a minute must also beentered. Additionally, as discussed previously, up to ten pre-assignedmethod parameters may be inputted into the system by following theprevious steps.

With respect to an analysis for which the run time is unknown, theoperator should enter the longest run time that is consideredappropriate for the analysis. After running the analysis, the actual runtime may be re-entered by simply depressing the “Select Method” buttonon the operator interface and scrolling through the parameters asdiscussed above.

Before accepting a method entered at steps 520-522, the system willcheck to confirm that the suppressor capacity is sufficient to completethe analysis within 40% of the total run time and that the power supplycan generate enough current to complete regeneration within 40% of thetotal run time. If the suppressor capacity is insufficient, program flowproceeds to step 523 where a message is generated on the system displaystating that there is a “cell [e.g. suppressor] type and methodmismatch.” The operator will either need to replace the suppressors withsuppressors having greater capacity, or change the method parameters byreducing the mobile phase concentration, flow rate or run time. Themethod parameters may be re-set by depressing the “Enter” button on theoperator interface at step 523 and program flow will revert back to step521, where the method parameters may be selected as previouslydiscussed. If the power supply does not have enough current to timelyregenerate the suppressor (i.e. within 40% of the run time), programflow proceeds to step 524 where a message is generated on the systemdisplay stating that “flow or conc. too high”. The operator will need toreduce the mobile phase concentration or the flow rate. As a generalrule, the product of the concentration in meq./L and the flowrate inML/min. should be equal or less than 22.5 (conc.×flowrate<22.5). In anyevent, the operator may change the parameters relating to mobile phaseconcentration or the flowrate by depressing the “Enter” button on theoperator interface at step 524 and program flow will revert back to step521, where the method parameters may be selected as discussed.

Based on the foregoing discussion, one skilled in the art willappreciate that the system software will not allow method parametersthat exceed 40% of the capacity of the individual suppressors. Thislimit is to ensure that, regardless of when a sample injection isreceived by the suppressor system, an individual suppressor will neverachieve full exhaustion. For example, a suppressor could have mobilephase flowing through it for almost the entire analysis time beforereceiving a sample injection. Thus, in a “worst case” scenario, thesuppressor could, at most, be nearly 40% exhausted before the sampleinjection reaches the suppressor. The suppressor, which is 40% exhaustedwhen it finally receives a sample injection, will nonetheless still have60% of its suppression capacity. Because the system does not acceptoperating parameters that will exceed 40% of an individual suppressor'scapacity, the suppressor will be able to complete the analysis withabout 20% of its capacity still remaining. Thus, the suppressor shouldtheoretically never exceed 80% exhaustion in the system. As thoseskilled in the art will appreciate, in the above-described system, themaximum useful cell capacity is actually 80% of label capacity.

The system further has a variety of pre-programmed sub-routines relatingto system errors. For example, if the voltage across a suppressorexceeds a pre-assigned value at any time during regeneration, an errormessage is generated on the system display at steps 530, 530 a or 530 b,depending on what mode of operation the system is in when the erroroccurs. This message will indicate which of the suppressors in thesystem has failed, and the system will automatically go into a standbymode. Such an error indicates that there is too much resistance in thesuppressor to achieve regeneration within 40% of the sample run time,and the suppressor should be checked. The upper pre-assigned voltagelimit is the upper voltage limit of the system power supply. Similarly,if the voltage across a suppressor is below a pre-assigned value at anytime during regeneration, an error message is generated on the systemdisplay at step 531. Such an error indicates that a short circuit hasoccurred between the electrodes. In any event, once the voltage problemis corrected, the system power is cycled and program flow reverts backto step 500. Alternatively, the system may be programmed such that, oncethe voltage problem is eliminated, the program flow reverts back toentry point B by depressing the “Enter” button on the operatorinterface.

Another error sub-routine is triggered if the cover of the suppressorcompartment on the front panel of the unit is open. In such an event,system operation is interrupted and a message is generated on the systemdisplay at step 540. A flashing light on the operator interface istriggered as well. If the cover is closed within a pre-assigned periodof time, the system will resume operation. If, however, the cover is notclosed before passage of this pre-assigned period of time, program flowthen proceeds to step 541. Once the cover is closed, program flow thenproceeds to entry point A. Alternatively, system operation may beaborted when the cover is open by pressing the “Enter” button at step540, and program flow proceeds to step 541.

Yet another error sub-routine is triggered if the HPLC pump in thesystem is either off when the system is powered-up or if the pump shutsdown during system operation. In either event, the system goes into astandby mode and a message is generated on the system display at step550. Once the problem is corrected, program flow proceeds to eitherentry point A (if the problem occurred while the system was running) orto entry point B (if the system was powered-up with the pump off).

The system is also capable of Remote Out-Put via “Remote Out” pinspreferably positioned on the rear panel of the operator interface. Thesystem will send a “Not Ready” signal to external devices such as anautomatic sample injection system when the system is not ready toreceive a sample injection. The system sends the “Not Ready” signal toexternal devices until the system is ready to receive a sampleinjection, which is at step 504. The “Not Ready” signal can also serveas a safeguard if a system failure occurs. Thus, if any of the errorsequences discussed above is triggered, a “Not Ready” signal istransmitted to peripheral devices. Additionally, when the system is inthe “Full Regen” mode and both suppressors are thus being regenerated, a“Not Ready” signal is likewise sent to external devices.

Conversely, the system is also capable of receiving Remote In-Put via“Remote In” pins preferably positioned on the rear panel of the operatorinterface. The system may accept an error signal from other externaldevices. When it receives such an error signal, the system willautomatically go into a standby mode.

The preferred columns for the suppressors used in the system are made ofa clear, cylindrical shaped, polymethylenepentane material. Theelectrode and column end fittings comprise a unitary piece. A sinteredfrit made from an alloy of PEEK and TEFLON is pressed fitted into theend fitting. Such a column is depicted at p. A0000065 of the Appendix.

The above described suppressor unit is preferably used in combinationwith other external devices in a chromatography system. The otherexternal devices suitable for use with the self-contained suppressorunit discussed above include an HPLC pump capable of low-pulsationsolvent delivery with flowrates preferably ranging from 0.01 mL/min. to10 mL/min. A preferred pump is the ALLTECH Model 526 or Model 426 HPLCpump. Also included in the chromatography system is a detector capableof measuring the analyte. A preferred detector is the ALLTECH Model 550conductivity detector with a temperature controlled cell compartmentadjustable from ambient to 60° C., which eliminates thermally-inducedbaseline noise and drift. Also included is a strip chart recorder ordata system capable of accepting analog voltage data. An autosampler ormanual injection valve for sample introduction. An ion chromatographycolumn capable of separating the species of interest. And, optionally, aguard column packed with material similar to the packing in theanalytical column.

As can be ascertained from the foregoing discussion, some of thebenefits associated with Applicant's system and method are that noseparate regenerant reagents or pumps are required and no chemical waste(other than the detector effluent generated on any IC system) iscreated. The system can be used without fragile membranes and willtolerate high backpressures for greater reliability than membrane-baseddevices. The system is furthermore compatible with electroactive eluantsand organic solvents and operates equally well with all common SICeluants overcoming the drawbacks associated with prior self-regeneratingsuppressors.

2. Alternate Valve Schemes

With reference to FIGS. 5C and 8D, an alternative 10-port switchingvalve scheme is depicted. In FIG. 8C, suppressor 212 is the activesuppressor as described previously. Before suppressor 212 is exhausted,the analytical column effluent is re-routed as depicted in FIG. 8D. InFIG. 8D, the suppressor 214 is the active suppressor and the detectoreffluent is routed from the detector 218 through the 10-port switchingvalve 210 to suppressor 212 to regenerate the suppressor 212 byelectrolysis of the detector effluent as previously described. As thoseskilled in the art will appreciate, to regenerate the cation exchangeresin in either suppressors 212 or 214, it is critical that the anode islocated at an inlet (upstream) side of the suppressor.

Valve schemes other than the 10-port switching valve described above mayalso be used in the present invention. With reference to FIGS. 9A and9B, a 6-port valve 240 may be used. In FIG. 9A, the chromatographyeffluent is routed from analytical column 208 through 6-port switchingvalve 240 to suppressor column 212 and to the detector 218 where thesample ions are detected. With reference to FIG. 9B, when the column 212is exhausted and needs to be regenerated, the 6-port switching valve 240is switched so the column effluent is routed from analytical column 208to a packed bed suppressor 242 with strong cation exchange resin in thehydrogen form (i.e., during anion analysis). The cation exchange resinmay be as previously described. In the packed bed suppressor 242, theanalytical column effluent (aqueous sodium hydroxide or aqueous sodiumcarbonate/bicarbonate) is converted to water or carbonic acid. Thepacked bed suppressor effluent is then routed through the detector 218to the exhausted column 212, where the water in the packed bedsuppressor effluent feeds the electrolysis at column 212 to regeneratethe suppressor column 212.

In this scheme, only one suppressor column 212 is used for the analysis.The electrochemical regeneration of the suppressor column 212 can bedone between injections, after each injection before the sample anionselute from the analytical column 208, or whenever necessary. The packingmaterial in the packed bed suppressor 242 may be coated with dye toprovide color indication of its condition. The packed bed suppressor 242may need to be replaced only once a month or less depending on its totalcapacity. Since the packed bed suppressor 242 is not used during thechromatographic analysis, its size is not limited.

In another aspect of this invention and as depicted in FIGS. 10A and10B, the system may use a 4-port switching valve 246. In FIG. 10A, theanalytical column effluent is routed from the analytical column 820through 4-port switching valve 246 to suppressor column 212. Thesuppressor effluent is then routed to the detector 218 (where the sampleanions are detected) and then to waste. When the suppressor column 212is exhausted and needs to be regenerated, the 4-port switching valve 246is switched so that the analytical column effluent is routed throughpacked bed suppressor 242 with strong cation exchange resin in thehydrogen form. The analytical column effluent is converted to water orcarbonic acid in the suppressor 242. The packed bed suppressor effluentis then routed through 4-port switching valve 246 to column 212 (seeFIG. 10B). The water in the packed bed suppressor effluent feeds theelectrolysis in column 212 to regenerate the column 212 as previouslydescribed. A disadvantage of this design compared to the 6-portswitching valve 240 or the 10-port switching valve 210 configurations isthat the gases generated during electrolysis will pass through thedetector 218 before going to waste. Gas bubbles may thus become trappedinside the detector 218, creating extreme baseline noise.

3. Suppressor for Cation Analysis

As those skilled in the art will appreciate, if the polarity of thepreviously described suppressor columns are reversed and thechromatography packing materials are changed from cation exchangepacking material to anion exchange packing material, the same systemconfigurations as previously described may be used as cationsuppressors. In cation analysis, the eluant is usually a solution of anacid such as hydrochloric acid, nitric acid, diaminoproprionic acidhydrochloride, or methanesulfonic acid. The anion exchange packingmaterial may be either anion exchange resins or membranes impregnatedwith anion exchange particles. Preferred anion exchange packingmaterials include primary, secondary, tertiary, or quaternary aminefunctionalized inorganic or organic particles. The most preferred anionexchange packing materials comprise quaternary amine functionalizedinorganic or organic particles.

In cation analysis, the following reactions take place in the suppressorcolumn (where hydrochloric acid is the eluant and the anion exchangematerial comprises a quaternary amine functionalized particle):

-   -   1) Eluant: HCl+Resin−NH₄ ⁺OH⁻→Resin−NH₄ ⁺Cl⁻I−H₂O    -   2) Analyte: XCl+Resin−NH₄ ⁺OH⁻→Resin−NH₄ ⁺Cl⁻+XOH        -   where X=cations (Na, K, Li, Mg, Ca, etc.)

To regenerate the suppressor column in cation analysis, the position ofthe anode and cathode is opposite of that for anion analysis (i.e., thecathode is placed on the side of the suppressor where the detectoreffluent enters the suppressor column). During electrolysis, thefollowing reaction takes place at the cathode:2H₂O+2e ⁻→H₂+20H⁻

The released hydroxide ions are routed through the column to convert thechloride form resin (exhausted anion exchange material) back to thehydroxide form according to the following reaction:Resin−NH₄ ⁺Cl⁻+OH⁻→Resin−NH₄ ⁺OH⁻+Cl⁻

The various valve schemes previously described can also be used forcation analysis.

In yet another embodiment of the present invention, the need forswitching between suppressors may be eliminated altogether. Instead ofrouting the detector effluent through a switching valve to regeneratethe non-active suppressor while the active suppressor is in use, theeluant itself may be used to regenerate the exhausted suppressor. Inthis embodiment, the aqueous eluant is flowed through the separatorcolumn to the exhausted (or partially exhausted) suppressor column.Depending on whether cation or anion analysis was just conducted, eitherhydroxide (cation analysis) or hydronium (anion analysis) ions aregenerated at the upstream electrode. The hydroxide or hydronium ions arethen flowed through the suppressor to convert the suppressor back toeither its hydroxide or hydronium form. The advantage to this embodimentis that it eliminates the need for two suppressor columns and theassociated switching valves for switching between suppressors. As oneskilled in the art will recognize, however, in this embodiment theanalyses will be interrupted while regenerating the suppressor.Moreover, the sample counter-ions in the eluant will compete with eitherthe hydronium or hydroxide ions in the suppressor column, and,therefore, total conversion of the exhausted suppressor back to eitherthe hydroxide or hydronium form will not be achieved. However, bycontrolling current and eluant flow, hydronium or hydroxide conversioncan be favored over the sample counter-ions. Although primary adaptedfor use in a one suppressor system the foregoing method of regeneratinga suppressor may also be used to regenerate exhausted suppressors in asystem that uses two or more suppressors.

According to yet another embodiment of the present invention, a membranesuppressor is provided. With reference to FIG. 24, a housing 300 isprovided comprising a first effluent flow channel 301 defined by amembrane 301 a. The membrane 301 a is preferably made of Nafion™, whichis a semi-permeable plastic material that has been functionalized tocontain exchangeable ion sites (not shown) as previously describedherein. Annular electrodes 302 and 303 are positioned at the upstreamand downstream ends, respectively, of the housing 300. The electrodesmay be as previously described. Reinforcing the membrane 301 a is ionexchange packing material 309. Aside from reinforcing the membrane 301 ato the outward pressure generated by the fluid flowing through channel301, the packing material 309 also completes a circuit formed byelectrodes 302 and 303, power source 306 and ion exchange packingmaterial 309. The ion exchange packing material 309 preferably comprisesthe same functional groups as the functionalized membrane 301 a.According to this embodiment, the suppressor may function as acontinuously regenerated membrane suppressor, and will be discussedspecifically in reference to anion analysis using an aqueous sodiumhydroxide eluant. However, as will be appreciated by those skilled inthe art, this embodiment may be easily adapted for cation analysis aswell as for use with other inorganic or organic eluants.

With reference to FIG. 24, the membrane 301 a preferably comprisesexchangeable hydronium ions. Similarly, the reinforcing ion exchangepacking material 309 preferably comprises exchangeable hydronium ions.The sample anions and eluant (not shown) are flowed through the firsteffluent flow channel 301 wherein the counter-ions (Na⁺ in this example)of the sample anions displace the hydronium ions on the membrane 301 a.Displaced hydronium ions combine with the sample anions to form thehighly conductive acids of the sample anions. Similarly, displacedhydronium ions combine with the eluant co-ions of the sample ions (e.g.,OH⁻ in this example) to form the less conductive water. The sampleanions (in their acid form) and water are flowed to the detector wherethe sample ions are detected. The detector effluent or another externalsource of water-containing solution is then routed through a secondeffluent channel 310 while power source 306 is turned on. An electriccurrent across the ion exchange packing material 309 is generated. Theupstream electrode 303 functions as the anode and the water in thedetector effluent is electrolyzed to yield, among other things,hydronium ions. The hydronium ions may then migrate through the packingmaterial 309 to the membrane 301 a where the hydronium ions displace thesodium ions on the membrane 301 a. By keeping the power supply 306turned on, a continuous supply of hydronium ions may be generated atelectrode 303 and continuously supplied to membrane 301 a therebymaintaining the membrane 301 a in a suppressing form indefinitely. Asthose skilled in the art will appreciate, the housing 300 may betube-shaped such that the packing material 309 is concentric with themembrane 301 a. Alternatively, the housing may be rectangular shapedsuch that the packing material 309 is adjacent the planar shapedmembranes 301 a on the side of the membrane opposite channel 301.Additionally, packing material 309 may also be positioned in channel 301for increased suppressor capacity and for further supporting membrane301 a to prevent membrane rupture.

4. High Purity Eluant Chromatography

The column illustrated in FIG. 1 can also be advantageously employed ina method and apparatus for generating a high purity eluant. Withreference to FIG. 11A, a deionized water source 100 is provided. A pump102 as previously described is connected to water source 100. Downstreamfrom pump 102 is a sample injector 104. Downstream from the sampleinjector 104 are three columns 112, 120 and 122, which are arranged inseries. Column 112 is preferably constructed as illustrated in FIG. 1.An electrical power source 116 is connected to column 112. Column 120 isan analytical (e.g. chromatography) column packed with chromatographypacking material (not shown). Column 122 is also preferably constructedas illustrated in FIG. 1, and is adapted for use as a solid-phasechemical suppressor in this embodiment. Located downstream from columns112, 120 and 122 is a conductivity detector 118, which is as previouslydescribed. Finally, downstream from detector 118, is a backpressurevalve 144 and an ion exchange bed 146, which are also as previouslydescribed.

For anion analysis, column 112 is adapted for use as an eluantgenerating source and is packed with cation exchange packing material(not shown). The packing material preferably comprises exchangeablesodium ions. Column 120 is packed with anion exchange packing material(not shown) which is selected as previously described. Finally, column122 is packed with cation exchange packing material (not shown)comprising exchangeable hydronium ions, and is selected as previouslydescribed. The electric power source 116 is connected to an anode (notshown) which is positioned at the upstream end of column 112, and acathode (not shown) which is positioned at the downstream end of thecolumn 112. A high purity eluant for anion analysis is generated asfollows.

The water-containing eluant is routed through eluant generating column112. Power source 116 is turned on thereby generating an electriccurrent sufficient to electrolyze water across the sodium form cationexchange packing material (not shown) in column 112. At the anode (notshown), which is located at the upstream end of column 112, thewater-containing eluant undergoes electrolysis thereby generatinghydronium ions as previously described. At the cathode (not shown),which is located at the downstream end of column 112, the electrolysisof the water-containing eluant generates hydroxide ions as previouslydescribed. The hydronium ions generated at the upstream end of thecolumn 112 flow across the sodium form cation exchange packing materialand displace the sodium ions. The released sodium ions combine with thehydroxide ions generated at the downstream end of the column 112 to forma high purity sodium hydroxide eluant.

The sample anions (which may be injected either before or after eluantgenerating column 112), and the high purity sodium hydroxide eluant arethen routed through analytical column 120, where the sample anions arethen separated. The analytical column effluent is then routed fromcolumn 120 to solid phase chemical suppressor 122. The sample anions areconverted to their highly conductive acids by exchanging theircounterions for the hydronium ions on the hydronium form cation exchangepacking material in suppressor 122. Similarly, the sodium hydroxideeluant is converted to relatively non-conductive water by exchanging itssodium ions for the hydronium ions on the hydronium-form cation exchangematerial in suppressor 122. The suppressor effluent is then routed fromsuppressor 122 to detector 118 where the sample anions are detected. Thedetector effluent is then routed through back-pressure regulator 144 toion exchange bed 146. For anion analysis, ion exchange bed 146 comprisesanion exchange packing material comprising exchangeable hydroxide ions,and is selected as previously described. The sample anions displace thehydroxide ions in the ion exchange bed 146. The released hydroxide ionscombine with the hydronium counterions of the sample anions to formwater. The water may then be routed back to water source 100.

As those skilled in the art will appreciate, in addition to generating ahigh purity eluant, the foregoing method can suitably be used in amethod of gradient elution chromatography by controlling the amount ofsodium hydroxide eluant generated in column 112. The higher the currentin column 112, the greater the concentration of sodium hydroxide thatwill be generated in column 112.

When columns 112 and 122 are exhausted, i.e. the ion exchange packingmaterial is converted to the hydronium form and sodium form,respectively, they may be regenerated, either on-line or off-line.On-line regeneration may be accomplished according to the followingsteps. With reference to FIG. 11B, the water-containing eluant isrerouted and routed to column 122. An electrical power source 123 isprovided. The power-source 123 is connected to an anode (not shown),which is positioned at the upstream end of column 122, and a cathode(not shown) which is positioned at the downstream end of column 122. Thewater-containing eluant enters column 122 at the anode (not shown) endof column 122. Power source 123 is turned on thereby generating anelectric current sufficient to electrolyze water across the cationexchange packing material (now in the sodium form) in column 122.Hydronium ions are generated at the anode end of column 122 by theelectrolysis of the water-containing eluant as previously described.Also, hydroxide ions are generated at the cathode end of the column 122by the electrolysis of water as previously described. The hydronium ionsare routed across the sodium form cation exchange material in column 122and displace the sodium ions thereby converting the cation exchangeresin back to the hydronium form. The released sodium ions and theexcess hydronium ions generated at the anode end of column 122 combinewith the hydroxide ions generated at the cathode end of column 122 toform a high purity sodium hydroxide and water eluant.

The high purity sodium hydroxide eluant (as well as any sample anions tobe detected) is routed through analytical column 120, where any sampleanions are separated as previously discussed, and then to column 112.The exhausted cation exchange packing material in column 112 is inhydronium form. The hydronium ions on the exhausted cation exchangematerial in column 112 are displaced by sodium ions in the sodiumhydroxide eluant thereby regenerating the cation exchange packingmaterial back to its sodium form. The released hydronium ions combinewith the hydroxide ions in the eluant to form relatively lowconductivity water. The water (and sample anions) may then be routed toa detector (not shown) where the sample anions are detected. Thedetector effluent may then be routed through an ion exchange bed (notshown) where the sample anions are retained and hydroxide ions arereleased and combine with the hydronium counterions of the sample anionsto form water as previously described. The water may then be routed tothe water source 100.

The foregoing method and apparatus can also be used for generating ahigh purity eluent for cation analysis, In this embodiment, the column112 is packed with anion exchange packing material, preferablycomprising exchangeable chloride ions. Column 120 is packed with cationexchange packing material preferably comprising exchangeable hydroniumions, and suppressor column 122 is packed with anion exchange packingmaterial comprising exchangeable hydroxide ions.

FIG. 11C is a schematic of an alternative method and apparatus forgenerating a high purity eluant and capable of a gradient. In thisembodiment, the eluant generating column 113 may comprise a disposablecartridge packed with either anion or cation exchange packing materialas previously described with respect to eluant generating column 112(see FIG. 11 a and accompanying text in specification). Also, the sampleis injected downstream from the eluant generating column 113.

In yet another embodiment of the present invention, salt gradients maybe achieved using the principles of the present invention. For example,two columns as illustrated in FIG. 1 may be placed in series; one columnpacked with cation exchange packing material (i.e., the cation column)and the other column packed with anion exchange packing material (i.e.,the anion column). Eluant is flowed through the columns while a currentis applied in these columns as previously discussed. Hydronium ions aregenerated at the upstream electrode of the cation column and are flowedthrough the cation column so as to replace the cations in the cationcolumn. Similarly, hydroxide ions are generated at the upstreamelectrode of the anion column and are flowed through the anion column soas to replace the anions in the anion column. The cations released fromthe cation column and the anions released from the anion column combineto form a salt. Thus, for example, a relatively pure salt gradient ofsodium chloride may be generated by packing the cation column withexchangeable sodium ions and packing the anion column with exchangeablechloride ions. As those skilled in the art will appreciate, a sodiumchloride gradient is desirable for separating species such as protein,which have somewhat neutral pH and relatively high ionic strength. Also,when exhausted, these cation and anion columns may be regenerated aspreviously discussed.

5. Combination with Hydrophobic Packing Material (HydrophobicSuppressor)

The column illustrated in FIG. 1 can also be used with a hydrophobicsuppressor column, which is particularly useful in methods of inorganicanion analysis using an organic anion as the eluant. With reference toFIG. 12A, the sample anions and the eluant (which comprises an organicanion) are routed through analytical column 8. The analytical column 8is packed with anion exchange packing material (not shown) as previouslydescribed. The sample anions are thus separated in the analytical column8 according to methods known by those skilled in the art.

The analytical column effluent is then routed to suppressor column 12,which is preferably constructed as illustrated in FIG. 1 and which ispacked with cation exchange packing material (for anion analysis). Thecation exchange packing material preferably comprises exchangeablehydronium ions (not shown) as previously described. The organic anioneluant is converted to its organic acid form by ion exchange with thecation exchange packing material in suppressor column 12. Similarly, thesample anions are converted to their highly conductive acid form by ionexchange with the cation exchange packing material in column 12.

Thus, two of the ion-exchange reactions that take place in thesuppressor column 12 are:

-   -   1) Eluant: Na+−Organic Anion-+Resin-SO₃ ⁻H⁺→Resin-SO₃        ⁻Na⁺+Organic Acid    -   2) Analyte: NaX+Resin-SO₃ ⁻H⁺→Resin-SO₃ ⁻Na⁺+HX    -   where X=anions (Cl, NO₂, Br, etc.)

The suppressor column effluent is then routed through a hydrophobicsuppressor 50 b, which is packed with organic or inorganicreversed-phase packing material (not shown), and preferably with organicreversed phase packing material. The preferred reversed-phase packingmaterial comprises polystyrene divinyl benzene copolymer. The organicacid eluant is adsorbed on the packing material in column 50 b andretained. The inorganic sample anions (which are in their acid form) arenot adsorbed by the hydrophobic suppressor 50 b and are routed toconductivity detector 18 as high conductivity acids in a stream of waterwhere they are detected. This device significantly increases signal tonoise ratios, just as other suppressors do, but may be used with a muchwider range of eluants, columns and methods.

The combination of the column of the present invention with ahydrophobic suppressor can be used to construct continuously-regenerablehydrophobic suppressor units 52 a and 52 b as illustrated in FIGS. 12Aand 12B.

The system depicted in FIGS. 12A and 12B will function properly untileither the cation exchange packing material in columns 12 or 14 becomeexhausted (converted to the sodium form) or until the capacity of thehydrophobic suppressors 50 a or 50 b to adsorb the eluant organic acidis exceeded. The relative bed sizes for the suppressor columns 12 and 14and the hydrophobic suppressors 50 a and 50 b are preferably chosen sothat the capacity of the suppressor columns 12 and 14 are exceededbefore that of the hydrophobic suppressors 50 a or 50 b.

With reference to FIGS. 12A and 12B, two such hydrophobic suppressorunits 52 a and 52 b, respectively, may be used in any of the valvearrangements previously described. Before the first hydrophobicsuppressor unit 52 a is exhausted, the valve 10 is switched (see FIG.12B) and the detector effluent is re-routed through the exhaustedhydrophobic suppressor unit 52 b. The detector effluent contains thesample inorganic anions in their acid form and water. The water in thedetector effluent is used to feed electrolysis for regenerating theexhausted hydrophobic suppressor unit 52 b. This is accomplished asfollows.

The detector effluent is routed to suppressor column 12 and power source16 b is turned on to generate an electric current sufficient for theelectrolysis of water across the exhausted cation exchange packingmaterial in column 12. The anode is located at the upstream end of thesuppressor column 12. Hydronium ions are thus generated at the upstreamside of suppressor column 12 as previously described. The hydronium ionsare routed through column 12 and displace the sample and eluantcounterions on the exhausted cation exchange packing material therebyregenerating the packing material. The hydroxide ions generated at thecathode, which is located at the downstream end of the suppressor column12, combine with the released sample and eluant counterions from thecolumn 12 to form their hydroxides.

These hydroxides are then routed through the hydrophobic suppressor 50 bbefore going to waste. It is well known that organic acids in theirionized state are very poorly adsorbed by hydrophobic packing materials.Thus, as the hydroxides are routed through the hydrophobic suppressor 50b, the strongly adsorbed organic acids are converted back to theirweakly adsorbed ionized salts causing them to desorb from thehydrophobic suppressor 50 b. The hydrophobic suppressor effluent is thenrouted to waste through 10-port valve switch 210. In this way, both thehydrophobic suppressor 50 b and the suppressor column 12 aresimultaneously regenerated.

A similar configuration can be envisioned for hydrophobic suppression incation analysis, except that the polarity of the suppressor columns 12and 14 are reversed, the suppressor columns are packed with anionexchange packing material comprising exchangeable hydroxide ions. Thesame hydrophobic suppressor packing material as previously described foranion analysis, however, may be used for cation analysis as well.

6. Other Applications

As those skilled in the art will readily appreciate based on theforegoing disclosure, the columns and methods of the present inventioncan be used in a variety of other applications as well. For example, thecolumn illustrated in FIG. 1 can be used as a sample pretreatment deviceto reduce the pH of basic samples or to increase pH of acidic samples.The columns of the present invention can be packed with cation exchangepacking material in the hydrogen form as previously described and can beused to reduce sample pH, removing hydroxide or carbonate. When thecation exchange packing is exhausted, it can be electrochemicallyregenerated as previously described. Conversely, the columns of thepresent invention can be packed with anion exchange packing material aspreviously described to increase sample pH or remove hydrogen ions. Whenthe anion exchange packing material is exhausted, it can beelectrochemically regenerated as previously described.

The column illustrated in FIG. 1 can also be used for other post columnreactions that are pH-dependent. For example, the columns of the presentinvention can also be used to regenerate solid phase reagent (SPR)suppressors. In SPR, an aqueous suspension of submicron size resin isadded post-column to the eluant stream to chemically suppress theeluant. U.S. Pat. No. 5,149,661 provides a detailed discussion of SPR,the disclosure of which is fully incorporated by reference herein. Byusing the column and methods of the present invention to performelectrolysis on the detector effluent, the released hydrogen orhydroxide ions can electrochemically regenerate the reagent and it canbe recirculated on-line.

The column illustrated in FIG. 1 may also be adapted as apreconcentration device for ion analysis. The column may be packed withany of the chromatography packing materials previously described.Samples containing components with a strong attraction to the chosenpacking may be routed through the column where they will be retained onthe packing contained therein. Thereafter, water may be routed throughthe column and power supplied to elute the sample as previouslydescribed. Very large volumes of dilute samples may be passed throughthe column. The retained sample mass may be electrochemically eluted ina much smaller volume and at a much higher concentration greatly aidingsubsequent separation and/or detection by, for example, chromatography,atomic adsorption, ICP, or mass spectrometry.

As those skilled in the art will appreciate based on the foregoingdisclosure, the apparatuses and methods of the present invention arebased on electrochemically modifying the mobile phase (e.g., the eluant)to modify the retention of a compound or species on the stationary phase(e.g., the chromatography material). Thus, the inventions andapparatuses of the present invention are not limited to the use ofwater-containing eluants. Indeed, any eluant that can beelectrochemically modified is contemplated for use in the methods andapparatuses of the present invention. For instance, eluants containingnon-aqueous species are also suitable for use in the present invention.Suitable non-aqueous species include alkyl, aromatic and olefinicalcohols, halogens, and thiols; aromatics in the presence of anucleophile; and organic acids, sulfuric and nitric acids (non-aqueous).However, where such non-aqueous species are used as the eluant,catalytic electrodes may be required. For example, where methyl alcohol(e.g., methanol) is used as the eluant, the following reactions takeplace at catalytically active rheuthenium cyanide electrodes:Anode: CH₃OH→2H⁺+CH₂O+2e ⁻Cathode: CH₂O+2e ⁻+H⁺→CH₃OH

Thus, as those skilled in the art will readily appreciate, byelectrochemically modifying the mobile phase (e.g., the eluant), theenvironment within the effluent flow channel may be modified whichthereby modifies the retention or affinity of the compound or speciesretained on the chromatography material in the effluent flow channel.

Finally, the methods and columns of the present invention have other,far-reaching applications outside of the chromatography field. Forexample, the methods and apparatuses of the present invention can beapplied to achieve a self-regenerating home water-softening system. Forexample, suitable chromatography packing materials (such as theion-exchange packing materials previously described) can be used toremove cations (e.g. hardness) from the water. Once the chromatographypacking material is exhausted, it can be regenerated as previouslydescribed by the electrolysis of water.

In order to illustrate certain embodiments of the present invention, thefollowing examples are provided. However, the examples should not beconstrued as to limiting the present invention, the scope of which isdefined by the appended claims and their equivalents.

EXAMPLE 1 Relationship Between Current, Regeneration or ElectrolysisTime, and Suppressor Capacity or Lifetime

FIG. 13 shows the relationship between the suppressor capacity of acolumn according to the present invention and the applied current duringregeneration. The voltage applied across the column during theelectrolysis is between 3-5 V. FIG. 14 shows the relationship betweensuppressor capacity and electrolysis regeneration time. These resultsshow that there is a linear relationship between electrolysis time,current, and suppressor capacity. Several of the curves in FIGS. 13 and14 have slopes greater than 1 demonstrating that the column of thepresent invention can be electrochemically regenerated in less time thanit takes to exhaust during use. This is required for cycling between twocolumns to work.

EXAMPLE 2 Reproducibility of the Regeneration Process

When three electrolysis regenerations were performed repeatedly on thesame column (10 minute electrolysis at 400 mAmps (¾″ diameter opening)),the following results were obtained:

Trial # Suppressor Capacity (min) 1 129 2 130 3 132

These results indicate that the regeneration process is consistent andreproducible.

EXAMPLE 3 Chromatography

FIG. 15 shows a chromatogram of anions obtained using the column of thepresent invention as a suppressor with 1″ diameter column packed withsulfonic-acid functionalized polystyrene-divinyl benzene cation exchangepacking material. The peaks are broad due to band broadening, resultingin loss of chromatographic efficiency. The band broadening is due to thelarge void volume in the suppressor column. FIG. 16 shows a chromatogramof anions obtained using 0.75 cm diameter cell packed with the samecation-exchange packing material. By reducing the bed diameter, bandbroadening is reduced.

EXAMPLE 4 Electroelution Ion Chromatography (Anion Analysis)

The following materials and conditions were used in this example:

-   Column: 6 mm×7.5 mm packed with anion-exchange functionalized    organic particles (trimethylammonium functionalized divinylbenzene    polymer)-   Eluant: Deionized water-   Flow rate: 1.0 mL/min.-   Detector: 350 conductivity detector-   Sample: Anion (fluoride, chloride)-   Electrolysis: Constant Voltage    Results:

FIG. 17 shows a separation of fluoride and chloride obtained on thiscolumn. The electrolysis was conducted at 28 V. A backpressure regulatorof 100 psi was installed at the detector outlet to reduce bubbleformation from the generation of O₂(g) and H₂(g) during theelectrolysis.

EXAMPLE 5 Cation Analysis Using Electrochemically Regenerated SuppressorColumn

The following materials and conditions were used:

-   Suppressor: Suppressor A—7.5 mm ID×0.9 mm thick Suppressor B—10 mm    ID×0.31″ thick-   Amount of-   packing: Packed with 0.40 grams anion exchange resin in hydroxide    form (trimethylammonium functionalized polystyrene-divinylbenzene    polymer) (Suppressors installed in 10-port Micro-Electric Actuator)-   Column: ALLTECH Universal Cation Column (polybutadiene-maleic acid    coated silica particles)-   Eluant: 3 mM Methane Sulfonic Acid-   Flow rate: 1.0 mL/min.-   Detector: 350 Conductivity Detector-   Sample: injections (A) lithium, (B) sodium, (C) ammonium, (D)    potassium, (E) magnesium, (F) calcium, 50 μL injection-   Electrolysis: Constant current at 79 mAmps    Results:

FIG. 18 shows a separation of cations obtained using a column accordingto the present invention as a suppressor for cation analysis.

EXAMPLE 6 Electroelution Ion Chromatography (Cation Analysis) with WaterContaining Eluant

The following materials and conditions were used:

-   -   Column: 30 mm×4.6 mm packed with ALLTECH Universal Cation Column        (polybutadiene-maleic acid coated silica particles)    -   Eluant: Deionized water containing 0.1 mm methane sulfonic acid    -   Flowrate: 11.0 mL/min    -   Detector: model 350 conductivity detector    -   Sample: lithium, sodium, ammonium, potassium        Results:

Deionized water was used as the eluant originally. Since a much longercolumn (30 mm-very high resistant) was used, the power supply (thispower supply has 36V upper limit) was not able to generate current forelectrolysis. No elution of cations was observed. A small amount ofmethane sulfonic acid was added to the water to reduce the resistance ofthe water eluant.

FIG. 19 shows the separation of the 4 cations with just the 0.1 mmmethane sulfonic acid and water as the eluant (power supply was turnedoff).

FIG. 20 shows the same separation with the power on. The peaks areeluted at much shorter time in FIG. 20. Thus, when the concentration ofthe hydronium ions is increased by generating an electric current in thecolumn, the sample cations are eluted faster. The electrolysis wasconducted at 28V. As depicted in FIG. 20, some baseline noise wasdetected in the analysis, which was caused by the bubbles formed duringelectrolysis of water. A backpressure regulator was not used in thisanalysis.

EXAMPLE 7 Gradient Electroelution Ion Chromatography (Anion Analysis)

The following materials and conditions were used in this example:

-   Column: 6 mm×7.5 mm column packed with anion exchange functionalized    organic particles (trimethylammonium functionalized divinylbenzene    polymer)-   Eluant: Deionized water-   Flowrate: 10 mL/min-   Detector: model 350 conductivity detector-   Sample: fluoride (5 ppm), chloride (10 ppm), nitrate (10 ppm); 50 μm    injection    Results:

FIG. 21 shows a separation of fluoride, chloride, and nitrate obtainedon this column with constant current electrolysis at 10 mAmp Theretention time for fluoride, chloride, and nitrate are 6.31, 8.79, and18.3 minutes, respectively. FIG. 22 shows the separation of the samecomponents on this column with constant current electrolysis at 16 mAmp.By increasing the current, the retention time for all anions is reduced.These results indicate that the amount of hydroxide ions produced duringelectrolysis is proportional to the amount of current used. By varyingthe electric current during the separation, the concentration of theeluant may be varied, thereby generating a gradient.

1. A chromatography apparatus for electroelution chromatography, saidapparatus comprising (a) a sample injector, (b) a housing comprising asample flow channel having an inlet and an outlet adapted to permitfluid flow through the housing, and chromatography packing materialdisposed in the sample flow channel capable of chromatographicallyseparating anions or cations, said sample flow channel inlet being influid communication with said sample injector, (c) first and secondelectrodes positioned so that at least a portion of the chromatographypacking material is disposed between the first and second electrodes,and (d) a detector in fluid communication with said sample flow channeloutlet.
 2. The apparatus of claim 1 in which said housing includingchromatography packing material is the sole housing includingchromatography packing material disposed between said sample injectorand detector.
 3. The apparatus of claim 1 further comprising an electricpower source connected to the first and second electrodes.
 4. Theapparatus of claim 1 wherein fluid flow through the housing is from oneof the first or second electrodes to the other.
 5. The apparatus ofclaim 1 further comprising an ion exchange bed disposed in a flow pathbetween said detector and sample injector.
 6. The apparatus of claim 5in which the chromatography packing material includes exchangeable ionsof one charge, positive or negative, and the ion exchange bed includesexchangeable ions of the opposite charge.
 7. A method of analysis ofsample ions in a liquid sample, said method comprising: (a) flowing aliquid sample including sample ions to be detected through a sample flowchannel packed with chromatography packing material and retaining thesample ions in the packing, (b) generating exchangeable ions of theretained sample ions by the electrolysis of water at an electrodepositioned at an upstream end of the sample flow channel, (c) moving thegenerated exchangeable ions through the chromatography packing materialthereby eluting the retained sample ions therefrom , and (d) flowing theeluted sample ions to a detector and detecting the eluted sample ions bythe detector.
 8. The method of claim 7 wherein the sample ions compriseanions, the chromatography packing material comprises anion exchangematerial, and the exchangeable ions comprise hydroxide ions.
 9. Themethod of claim 7 comprising the additional step of flowing effluentfrom the detector through an ion exchange bed comprising exchangeablehydroxide ions.
 10. The method of claim 9 comprising the additional stepof flowing the effluent from the ion exchange bed to a water-containingeluant source and to said sample flow channel.
 11. The method of claim 7wherein the sample ions comprise cations, the chromatography packingmaterial comprises cation exchange material, and the exchangeable ionscomprise hydronium ions.
 12. The method of claim 11 comprising theadditional step of flowing effluent from the detector through an ionexchange bed comprising exchangeable hydronium ions.
 13. The method ofclaim 11 comprising the additional step of flowing the effluent from theion exchange bed to a water-containing eluant source.